Methods and compositions comprising purified recombinant polypeptides

ABSTRACT

Purified recombinant polypeptides isolated from Chinese hamster ovary host cells, including antibodies, such as therapeutic antibodies, and methods of making and using such polypeptides are provided.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/065,693, filed Mar. 9, 2016, which is a continuation of InternationalApplication No. PCT/US2014/055387 having an international filing date ofSep. 12, 2014, which claims the benefit of priority of provisional U.S.Application No. 61/877,517 filed Sep. 13, 2013, each of which isincorporated by reference herein in its entirety for any purpose.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 1, 2018, is named2018-02-01_01146-0063-01US_sequence_listing.txt and is 34,888 bytes insize.

FIELD

Purified recombinant polypeptides isolated from Chinese hamster ovaryhost cells, including antibodies, such as therapeutic antibodies, andmethods of making and using such polypeptides are provided.

BACKGROUND

A number of drugs are on the market or in development for treatingasthma and other respiratory disorders. One of the targets for asthmatherapy is IL-13. IL-13 is a pleiotropic TH2 cytokine produced byactivated T cells, NKT cells, basophils, eosinophils, and mast cells,and it has been strongly implicated in the pathogenesis of asthma inpreclinical models. IL-13 antagonists, including anti-IL-13 antibodies,have previously been described. Certain such antibodies have also beendeveloped as human therapeutics. Recently, several studies have shownclinical activity of monoclonal antibodies against IL-13 in thetreatment of asthma (See, e.g., Corren et al., 2011, N. Engl. J. Med.365, 1088-1098; Gauvreau et al., 2011, Am. J. Respir. Crit. Care Med.183, 1007-1014; Ingram and Kraft, 2012, J. Allergy Clin. Immunol. 130,829-42; Webb, 2011, Nat Biotechnol 29, 860-863). Of these, lebrikizumab,a humanized IgG4 antibody that neutralizes IL-13 activity, improved lungfunction in asthmatics who were symptomatic despite treatment with, forthe majority, inhaled corticosteroids and a long-acting beta2-adrenergicreceptor agonist (Corren et al., 2011, N. Engl. J. Med. 365, 1088-1098).

In addition, IL-13 has been implicated in numerous other allergic andfibrotic disorders. For example, such diseases and/or conditionsmediated by IL13 include, but are not limited to, allergic asthma,non-allergic (intrinsic) asthma, allergic rhinitis, atopic dermatitis,allergic conjunctivitis, eczema, urticaria, food allergies, chronicobstructive pulmonary disease, ulcerative colitis, RSV infection,uveitis, scleroderma, and osteoporosis.

For recombinant biopharmaceutical proteins to be acceptable foradministration to human patients, it is important that residualimpurities resulting from the manufacture and purification process areremoved from the final biological product. These process componentsinclude culture medium proteins, immunoglobulin affinity ligands,viruses, endotoxin, DNA, and host cell proteins. These host cellimpurities include process-specific host cell proteins (HCPs), which areprocess-related impurities/contaminants in the biologics derived fromrecombinant DNA technology. While HCPs are typically present in thefinal drug substance in small quantities (in parts-per-million ornanograms per milligram of the intended recombinant protein), it isrecognized that HCPs are undesirable and their quantities should beminimized. For example, the U.S. Food and Drug Administration (FDA)requires that biopharmaceuticals intended for in vivo human use shouldbe as free as possible of extraneous impurities, and requires tests fordetection and quantitation of potential contaminants/impurities, such asHCPs.

Procedures for purification of proteins from cell debris initiallydepend on the site of expression of the protein. Some proteins aresecreted directly from the cell into the surrounding growth media;others are made intracellularly. For the latter proteins, the first stepof a purification process involves lysis of the cell, which can be doneby a variety of methods, including mechanical shear, osmotic shock, orenzymatic treatments. Such disruption releases the entire contents ofthe cell into the homogenate, and in addition produces subcellularfragments that are difficult to remove due to their small size. Theseare generally removed by centrifugation or by filtration. The sameproblem arises with directly secreted proteins due to the natural deathof cells and release of intracellular host cell proteins in the courseof the protein production run.

Once a solution containing the protein of interest is obtained, itsseparation from the other proteins produced by the cell is usuallyattempted using a combination of different chromatography techniques.Typically, these techniques separate mixtures of proteins on the basisof their charge, degree of hydrophobicity, or size. Several differentchromatography resins are available for each of these techniques,allowing accurate tailoring of the purification scheme to the particularprotein involved. The essence of each of these separation methods isthat proteins can be caused either to move at different rates down along column, achieving a physical separation that increases as they passfurther down the column, or to adhere selectively to the separationmedium, being then differentially eluted by different solvents. In somecases, the desired protein is separated from impurities when theimpurities specifically adhere to the column, and the protein ofinterest does not, that is, the protein of interest is present in the“flow-through.”

Ion-exchange chromatography, named for the exchangeable counterion, is aprocedure applicable to purification of ionizable molecules. Ionizedmolecules are separated on the basis of the non-specific electrostaticinteraction of their charged groups with oppositely charged moleculesattached to the solid phase support matrix, thereby retarding thoseionized molecules that interact more strongly with solid phase. The netcharge of each type of ionized molecule, and its affinity for thematrix, varies according to the number of charged groups, the charge ofeach group, and the nature of the molecules competing for interactionwith the charged solid phase matrix. These differences result inresolution of various molecule types by ion-exchange chromatography. Intypical protein purification using ion exchange chromatography, amixture of many proteins derived from a host cell, such as in mammaliancell culture, is applied to an ion-exchange column. After non-bindingmolecules are washed away, conditions are adjusted, such as by changingpH, counter ion concentration and the like in step- or gradient-mode, torelease from the solid phase a non-specifically retained or retardedionized protein of interest and separating it from proteins havingdifferent charge characteristics. Anion exchange chromatography involvescompetition of an anionic molecule of interest with the negative counterion for interaction with a positively charged molecule attached to thesolid phase matrix at the pH and under the conditions of a particularseparation process. By contrast, cation exchange chromatography involvescompetition of a cationic molecule of interest with the positive counterion for a negatively charged molecule attached to the solid phase matrixat the pH and under the conditions of a particular separation process.Mixed mode ion exchange chromatography (also referred to as multimodalion exchange chromatography) involves the use of a combination of cationand anion exchange chromatographic media in the same step. Inparticular, “mixed mode” refers to a solid phase support matrix to whichis covalently attached a mixture of cation exchange, anion exchange, andhydrophobic interaction moieties.

Hydroxyapatite chromatography of proteins involves the non-specificinteraction of the charged amino or carboxylate groups of a protein withoppositely charged groups on the hydroxyapatite, where the net charge ofthe hydroxyapatite and protein are controlled by the pH of the buffer.Elution is accomplished by displacing the non-specificprotein-hydroxyapatite pairing with ions such as Ca2+ or Mg2+.Negatively charged protein groups are displaced by negatively chargedcompounds, such as phosphates, thereby eluting a net-negatively chargedprotein.

Hydrophobic interaction chromatography (HIC) is typically used for thepurification and separation of molecules, such as proteins, based ondifferences in their surface hydrophobicity. Hydrophobic groups of aprotein interact non-specifically with hydrophobic groups coupled to thechromatography matrix. Differences in the number and nature of proteinsurface hydrophobic groups results in differential retardation ofproteins on a HIC column and, as a result, separation of proteins in amixture of proteins.

Affinity chromatography, which exploits a specific structurallydependent (i.e., spatially complementary) interaction between theprotein to be purified and an immobilized capture agent, is a standardpurification option for some proteins, such as antibodies. Protein A,for example, is a useful adsorbent for affinity chromatography ofproteins, such as antibodies, which contain an Fc region. Protein A is a41 kD cell wall protein from Staphylococcus aureas which binds with ahigh affinity (about 10⁻⁸M to human IgG) to the Fc region of antibodies.

Purification of recombinant polypeptides is typically performed usingbind and elute chromatography (B/E) or flow-through (F/T)chromatography. These are briefly described below.

Bind and Elute Chromatography (B/E): Under B/E chromatography theproduct is usually loaded to maximize dynamic binding capacity (DBC) tothe chromatography material and then wash and elution conditions areidentified such that maximum product purity is attained in the eluate.

Various B/E methods for use with protein A affinity chromatography,including various intermediate wash buffers, have been described. Forexample, U.S. Pat. Nos. 6,127,526 and 6,333,398 describe an intermediatewash step during Protein A chromatography using hydrophobicelectrolytes, e.g., tetramethylammonium chloride (TMAC) andtetraethylammonium chloride (TEAC), to remove the contaminants, but notthe immobilized Protein A or the protein of interest, bound to theProtein A column. U.S. Pat. No. 6,870,034 describes additional methodsand wash buffers for use with protein A affinity chromatography.

Flow Through Chromatography (F/T): Using F/T chromatography, loadconditions are identified where impurities strongly bind to thechromatography material while the product flows through. F/Tchromatography allows high load density for standard monoclonal antibodypreparations (MAbs).

In recombinant anti-IL13 MAb preparations and certain other recombinantpolypeptides produced in CHO cells, we identified an enzyme,phospholipase B-like 2, as a single CHOP species present in excess ofavailable antibodies in a total CHOP ELISA assay. As used herein, “PLB2”and “PLBL2” and “PLBD2” are used interchangeably and refer to the enzyme“phospholipase B-like 2” or its synonym, “phospholipase B-domain-like2”. Certain scientific publications on PLBL2 include Lakomek, K. et al.,BMC Structural Biology 9:56 (2009); Deuschi, et al., FEBS Lett580:5747-5752 (2006). PLBL2 is synthesized as a pre-pro-enzyme withparent MW of about 66,000. There is an initial leader sequence which isremoved and potential 6 mannose-6-phosphate (M-6-P) groups are addedduring post-translational modification. M-6-P is a targetingmodification that directs this enzyme to the lysosome via the M-6-Preceptor. PLBL2 contains 6 cysteines, two of which have freesulfhydrals, and four form disulfide bonds. In acidic environments,PLBL2 is further clipped into the N- and C-terminal fragments having32,000 and 45,000 MW, respectively. By analogy with other lysosomalenzymes, this cleavage is an activating step, allowing and access of thesubstrate to the active site.

There is about 80% PLBL2 amino acid sequence homology between hamsterand human forms of the enzyme. The enzyme activity is thought to be tocleave either fatty acid chain from the phospholipids that make up cellmembranes. There are other phospholipases with different substratecleavage specificities. Similar enzymatic activities exist inmicroorganisms, where they are often a virulence factor. Althoughmicroorganisms have a similar enzymatic activity, the protein generatingthis activity is different, and there is low sequence homology betweenmicrobial and mammalian PLBL2 enzymes. Phospholipases produce free fattyacids (FFA) as one product of the substrate hydrolysis. Free fatty acidsare themselves a potential immune-signaling factor. Dehydrogenationconverts FFA to arachadonic acid which potentially participates ininflammation cascades involving eicosanoids.

Having identified PLBL2 as a single HCP (CHOP) in recombinant anti-IL13MAb preparations and certain other recombinant polypeptides produced inCHO cells, we developed reagents, methods, and kits for the specific,sensitive, and quantitative determination of PLBL2 levels in anti-IL-13Mab preparations (and other recombinant polypeptide products) and atvarious stages of purification. These are briefly described in theExamples below and also in U.S. Provisional Patent Application Nos.61/877,503 and 61/991,228. In addition, there was the formidablechallenge of developing a large-scale, robust, and efficient process forthe purification of anti-IL13 MAb (and other recombinant polypeptideproducts) resulting in MAb of sufficient purity (including removal ofPLBL2) for human therapeutic use including late-stage clinical andcommercial use. The invention described herein meets certain of theabove-described needs and provides other benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

The invention is based, at least in part, on the development of improvedprocesses for the purification of recombinant polypeptides produced inChinese hamster ovary (CHO) cells that provide purified product withsubstantially reduced levels of hamster PLBL2. Recombinant polypeptidespurified according to the methods of the invention, includingtherapeutic antibodies such as an anti-IL13 antibody, may have reducedimmunogenicity when administered to human subjects.

Accordingly, in one aspect, compositions comprising an anti-IL13monoclonal antibody purified from CHO cells comprising the anti-IL13antibody and a residual amount of hamster PLBL2 are provided. In certainembodiments, the amount of hamster PLBL2 is less than 20 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 15 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 10ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than8 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 5 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 3 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 2 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is less than 1 ng/mg. In certain embodiments, the amount ofhamster PLBL2 is less than 0.5 ng/mg. In certain embodiments, the amountof hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mgand 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mgand 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and3 ng/mg, or between 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1ng/mg, or between the limit of assay quantitation (LOQ) and 1 ng/mg. Incertain embodiments, the anti-IL13 antibody comprises three heavy chainCDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 1, CDR-H2having the amino acid sequence of SEQ ID NO.: 2, and CDR-H3 having theamino acid sequence of SEQ ID NO.: 3, and three light chain CDRs, CDR-L1having the amino acid sequence of SEQ ID NO.: 4, CDR-L2 having the aminoacid sequence of SEQ ID NO.: 5, and CDR-L3 having the amino acidsequence of SEQ ID NO.: 6. In certain embodiments, the anti-IL13antibody comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO.: 7. In certain embodiments, the anti-IL13antibody comprises a light chain variable region having the amino acidsequence of SEQ ID NO.: 9. In certain embodiments, the anti-IL13antibody comprises a heavy chain having the amino acid sequence of SEQID NO.: 10. In certain embodiments, the anti-IL13 antibody comprises alight chain having the amino acid sequence of SEQ ID NO.: 14. In certainembodiments, the anti-IL13 antibody comprises a heavy chain variableregion having the amino acid sequence of SEQ ID NO.: 7 and a light chainvariable region having the amino acid sequence of SEQ ID NO.: 9. Incertain embodiments, the anti-IL13 antibody comprises a heavy chainhaving the amino acid sequence of SEQ ID NO.: 10 and a light chainhaving the amino acid sequence of SEQ ID NO.: 14. In certainembodiments, the amount of hamster PLBL2 in the composition isquantified using an immunoassay or a mass spectrometry assay. In certainembodiments, the immunoassay is a total Chinese hamster ovary proteinELISA or a hamster PLBL2 ELISA. In certain embodiments, the massspectrometry assay is LC-MS/MS.

In another aspect, anti-IL13 monoclonal antibody preparations isolatedand purified from CHO cells by a process comprising a hydrophobicinteraction chromatography (HIC) step are provided. In certainembodiments, the purified preparation comprises the anti-IL13 antibodyand a residual amount of hamster PLBL2. In certain embodiments, theamount of hamster PLBL2 is less than 20 ng/mg. In certain embodiments,the amount of hamster PLBL2 is less than 15 ng/mg. In certainembodiments, the amount of hamster PLBL2 is less than 10 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 8 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 5ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than3 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 2 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 1 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 0.5 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15ng/mg, or between 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mg and 8ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3ng/mg, or between 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1ng/mg, or between the limit of assay quantitation (LOQ) and 1 ng/mg. Incertain embodiments, the HIC step comprises PHENYL SEPHAROSE™ 6 FastFlow (High Sub) resin. In certain embodiments, the HIC step comprisesoperating a resin-containing column in flow-through mode. In certainembodiments, the HIC step comprises an equilibration buffer and a washbuffer, wherein each of the equilibration buffer and the wash buffercomprise 50 mM sodium acetate pH 5.0. In certain embodiments, theflow-through is monitored by absorbance at 280 nanometers and theflow-through is collected between 0.5 OD to 1.5 OD. In certainembodiments, the flow-through is collected for a maximum of 8 columnvolumes. In certain embodiments, the process further comprises anaffinity chromatography step. In certain embodiments, the affinitychromatography is protein A chromatography. In certain embodiments, theprocess further comprises an ion exchange chromatography step. Incertain embodiments, the ion exchange chromatography is anion exchangechromatography. In certain embodiments, the anti-IL13 antibody comprisesthree heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ IDNO.: 1, CDR-H2 having the amino acid sequence of SEQ ID NO.: 2, andCDR-H3 having the amino acid sequence of SEQ ID NO.: 3, and three lightchain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.: 4,CDR-L2 having the amino acid sequence of SEQ ID NO.: 5, and CDR-L3having the amino acid sequence of SEQ ID NO.: 6. In certain embodiments,the anti-IL13 antibody comprises a heavy chain variable region havingthe amino acid sequence of SEQ ID NO.: 7. In certain embodiments, theanti-IL13 antibody comprises a light chain variable region having theamino acid sequence of SEQ ID NO.: 9. In certain embodiments, theanti-IL13 antibody comprises a heavy chain having the amino acidsequence of SEQ ID NO.: 10. In certain embodiments, the anti-IL13antibody comprises a light chain having the amino acid sequence of SEQID NO.: 14. In certain embodiments, the anti-IL13 antibody comprises aheavy chain variable region having the amino acid sequence of SEQ IDNO.: 7 and a light chain variable region having the amino acid sequenceof SEQ ID NO.: 9. In certain embodiments, the anti-IL13 antibodycomprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10and a light chain having the amino acid sequence of SEQ ID NO.: 14. Incertain embodiments, the amount of hamster PLBL2 is quantified using animmunoassay or a mass spectrometry assay. In certain embodiments, theimmunoassay is a total Chinese hamster ovary protein ELISA or a hamsterPLBL2 ELISA. In certain embodiments, the mass spectrometry assay isLC-MS/MS.

In yet another aspect, purified anti-IL13 monoclonal antibodypreparations isolated from CHO cells are provided. In certainembodiments, the antibody preparation is purified by a processcomprising a first Protein A affinity chromatography step, a secondanion exchange chromatography step, and a third hydrophobic interactionchromatography (HIC) step thereby producing a purified preparation, Incertain embodiments, the purified preparation comprises the anti-IL13antibody and a residual amount of hamster PLBL2. In certain embodiments,the amount of hamster PLBL2 is less than 20 ng/mg. In certainembodiments, the amount of hamster PLBL2 is less than 15 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 10 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 8ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than5 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 3 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 2 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 1 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is less than 0.5 ng/mg. In certain embodiments, the amount ofhamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mgand 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mgand 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and3 ng/mg, or between 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1ng/mg, or between the limit of assay quantitation (LOQ) and 1 ng/mg. Incertain embodiments, the affinity chromatography step comprisesMABSELECT SURE™ resin, the anion exchange chromatography step comprisesQ SEPHAROSE™ Fast Flow, and the HIC step comprises PHENYL SEPHAROSE™ 6Fast Flow (high sub). In certain embodiments, the affinitychromatography step comprises operating a MABSELECT SURE™resin-containing column in bind-elute mode, the anion exchangechromatography step comprises operating a Q SEPHAROSE™ Fast Flowresin-containing column in bind-elute mode, and the HIC step comprisesoperating a PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin-containingcolumn in flow-through mode. In certain embodiments, the anti-IL13antibody comprises three heavy chain CDRs, CDR-H1 having the amino acidsequence of SEQ ID NO.: 1, CDR-H2 having the amino acid sequence of SEQID NO.: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO.: 3,and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQID NO.: 4, CDR-L2 having the amino acid sequence of SEQ ID NO.: 5, andCDR-L3 having the amino acid sequence of SEQ ID NO.: 6. In certainembodiments, the anti-IL13 antibody comprises a heavy chain variableregion having the amino acid sequence of SEQ ID NO.: 7. In certainembodiments, the anti-IL13 antibody comprises a light chain variableregion having the amino acid sequence of SEQ ID NO.: 9. In certainembodiments, the anti-IL13 antibody comprises a heavy chain having theamino acid sequence of SEQ ID NO.: 10. In certain embodiments, theanti-IL13 antibody comprises a light chain having the amino acidsequence of SEQ ID NO.: 14. In certain embodiments, the anti-IL13antibody comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO.: 7 and a light chain variable region having theamino acid sequence of SEQ ID NO.: 9. In certain embodiments, theanti-IL13 antibody comprises a heavy chain having the amino acidsequence of SEQ ID NO.: 10 and a light chain having the amino acidsequence of SEQ ID NO.: 14. In certain embodiments, the amount ofhamster PLBL2 is quantified using an immunoassay or a mass spectrometryassay. In certain embodiments, the immunoassay is a total Chinesehamster ovary protein ELISA or a hamster PLBL2 ELISA. In certainembodiments, the mass spectrometry assay is LC-MS/MS.

In still yet another aspect, methods of purifying a recombinantpolypeptide produced in CHO cells, wherein the method provides apurified preparation comprising the recombinant polypeptide and residualamount of hamster PLBL2 are provided. In certain embodiments, the amountof hamster PLBL2 is less than 20 ng/mg. In certain embodiments, theamount of hamster PLBL2 is less than 15 ng/mg. In certain embodiments,the amount of hamster PLBL2 is less than 10 ng/mg. In certainembodiments, the amount of hamster PLBL2 is less than 8 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 5 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 3ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than2 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 1 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 0.5 ng/mg. In certain embodiments, the amount of hamster PLBL2is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, orbetween 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mg and 8 ng/mg, orbetween 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, orbetween 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, orbetween the limit of assay quantitation (LOQ) and 1 ng/mg. In certainembodiments, the recombinant polypeptide is selected from a growthfactor, a cytokine, an antibody, an antibody fragment, and animmunoadhesin. In certain embodiments, the recombinant polypeptide is anantibody. In certain embodiments, the antibody is a humanized monoclonalantibody. In certain embodiments, the antibody is IgG1, or IgG2, orIgG3, or IgG4. In certain embodiments, the antibody is IgG1. In certainembodiments, the antibody is IgG2. In certain embodiments, the antibodyis IgG3. In certain embodiments, the antibody is IgG4. In certainembodiments, the methods comprise a hydrophobic interactionchromatography (HIC) step. In certain embodiments, the HIC stepcomprises PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin.

In certain embodiments of the above purification methods, the purifiedantibody is anti-IL13. In certain embodiments, the antibody islebrikizumab. In certain embodiments, the HIC step comprises operating aresin-containing column in flow-through mode. In certain embodiments,the HIC step comprises an equilibration buffer and a wash buffer,wherein each of the equilibration buffer and the wash buffer comprise 50mM sodium acetate pH 5.0. In certain embodiments, the flow-through ismonitored by absorbance at 280 nanometers and the flow-through iscollected between 0.5 OD to 1.5 OD. In certain embodiments, theflow-through is collected for a maximum of 8 column volumes. In certainembodiments, the methods further comprise an affinity chromatographystep. In certain embodiments, the affinity chromatography is protein Achromatography. In certain embodiments, the methods further comprise anion exchange chromatography step. In certain embodiments, the ionexchange chromatography is anion exchange chromatography. In certainembodiments, the methods comprise a first Protein A affinitychromatography step, a second anion exchange chromatography step, and athird hydrophobic interaction chromatography (HIC) step. In certainembodiments, the affinity chromatography step comprises MABSELECT SURE™resin, the anion exchange chromatography step comprises Q SEPHAROSE™Fast Flow, and the HIC step comprises PHENYL SEPHAROSE™ 6 Fast Flow(high sub). In certain embodiments, the affinity chromatography stepcomprises operating a MABSELECT SURE™ resin-containing column inbind-elute mode, the anion exchange chromatography step comprisesoperating a Q SEPHAROSE™ Fast Flow resin-containing column in bind-elutemode, and the HIC step comprises operating a PHENYL SEPHAROSE™ 6 FastFlow (High Sub) resin-containing column in flow-through mode. In certainembodiments, the amount of hamster PLBL2 is quantified using animmunoassay or a mass spectrometry assay. In certain embodiments, theimmunoassay is a total Chinese hamster ovary protein ELISA or a hamsterPLBL2 ELISA. In certain embodiments, the mass spectrometry assay isLC-MS/MS.

In certain embodiments of the above purification methods, the purifiedantibody is anti-Abeta. In certain embodiments, the anti-Abeta antibodyis crenezumab. In certain embodiments, the anti-Abeta antibody comprisesthree heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ IDNO.:23, CDR-H2 having the amino acid sequence of SEQ ID NO.:24, andCDR-H3 having the amino acid sequence of SEQ ID NO.:25, and three lightchain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.:26,CDR-L2 having the amino acid sequence of SEQ ID NO.:27, and CDR-L3having the amino acid sequence of SEQ ID NO.:28. In certain embodiments,the anti-Abeta antibody comprises a heavy chain variable region havingthe amino acid sequence of SEQ ID NO.:29. In certain embodiments, theanti-Abeta antibody comprises a light chain variable region having theamino acid sequence of SEQ ID NO.:30. In certain embodiments, theanti-Abeta antibody comprises a heavy chain variable region having theamino acid sequence of SEQ ID NO.:29 and a light chain variable regionhaving the amino acid sequence of SEQ ID NO.:30. In certain embodiments,the HIC step comprises operating a resin-containing column inflow-through mode. In certain embodiments, the HIC step comprises anequilibration buffer and a wash buffer, wherein each of theequilibration buffer and the wash buffer comprise 150 mM sodium acetatepH 5.0. In certain embodiments, the HIC step comprises an equilibrationbuffer and a wash buffer, wherein each of the equilibration buffer andthe wash buffer comprise 150 mM sodium acetate pH 4.0. In certainembodiments, the HIC step comprises an equilibration buffer and a washbuffer, wherein each of the equilibration buffer and the wash buffercomprise 150 mM sodium acetate, 240 mM sodium sulfate pH 4.0. In certainembodiments, the HIC step comprises an equilibration buffer and a washbuffer, wherein each of the equilibration buffer and the wash buffercomprise 150 mM sodium acetate, 240 mM sodium sulfate pH 5.0. In certainembodiments, the load density is 300 g/L. In certain embodiments, theload density is 100 g/L. In certain embodiments, the flow-through ismonitored by absorbance at 280 nanometers and the flow-through iscollected beginning at 0.5 OD and collection continues for 10 columnvolumes. In certain embodiments, the methods further comprise anaffinity chromatography step. In certain embodiments, the affinitychromatography is protein A chromatography. In certain embodiments, themethods further comprise a mixed mode chromatography step. In certainembodiments, the methods comprise a first Protein A affinitychromatography step, a second mixed mode chromatography step, and athird hydrophobic interaction chromatography (HIC) step. In certainembodiments, the affinity chromatography step comprises MABSELECT SURE™resin, the mixed mode chromatography step comprises CAPTO™ Adhere, andthe HIC step comprises PHENYL SEPHAROSE™ 6 Fast Flow (high sub). Incertain embodiments, the affinity chromatography step comprisesoperating a MABSELECT SURE™ resin-containing column in bind-elute mode,the mixed mode chromatography step comprises operating a CAPTO™ Adhereresin-containing column in flow-through mode, and the HIC step comprisesoperating a PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin-containingcolumn in flow-through mode. In certain embodiments, the amount ofhamster PLBL2 is quantified using an immunoassay or a mass spectrometryassay. In certain embodiments, the immunoassay is a total Chinesehamster ovary protein ELISA or a hamster PLBL2 ELISA. In certainembodiments, the mass spectrometry assay is LC-MS/MS.

In yet a further aspect of the above purification methods, the purifiedantibody is IgG1. In some embodiments, the antibody is anti-IL17 A/F. Insome embodiments, the anti-IL17 A/F antibody comprises three heavy chainCDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.:15, CDR-H2having the amino acid sequence of SEQ ID NO.:16, and CDR-H3 having theamino acid sequence of SEQ ID NO.:17, and three light chain CDRs, CDR-L1having the amino acid sequence of SEQ ID NO.:18, CDR-L2 having the aminoacid sequence of SEQ ID NO.:19 and CDR-L3 having the amino acid sequenceof SEQ ID NO.:20. In certain embodiments, the anti-IL17 A/F antibodycomprises a heavy chain variable region having the amino acid sequenceof SEQ ID NO.:21. In certain embodiments, the anti-IL17 A/F antibodycomprises a light chain variable region having the amino acid sequenceof SEQ ID NO.:22. In certain embodiments, the anti-IL17 A/F antibodycomprises a heavy chain variable region having the amino acid sequenceof SEQ ID NO.:21 and a light chain variable region having the amino acidsequence of SEQ ID NO.:22. In certain embodiments, the HICchromatography step comprises an equilibration buffer and a wash buffer,wherein each of the equilibration buffer and the wash buffer comprise 50mM sodium acetate pH 5.5. In certain embodiments, the flow-through ismonitored by absorbance at 280 nanometers and the flow-through iscollected beginning at 0.5 OD and for 10 column volumes. In certainembodiments, the methods further comprise an affinity chromatographystep. In certain embodiments, the affinity chromatography is protein Achromatography. In certain embodiments, the methods further comprise acation exchange chromatography step. In some embodiments, the methodscomprise a first Protein A affinity chromatography step and a secondcation exchange chromatography step prior to the hydrophobic interactionchromatography (HIC) step. In some embodiments, the affinitychromatography step comprises MABSELECT SURE™ resin, the cation exchangechromatography step comprises POROS 50 HS resin, and the HIC stepcomprises PHENYL SEPHAROSE™ 6 Fast Flow (high sub) resin. In someembodiments, the affinity chromatography step comprises operating aMABSELECT SURE™ resin-containing column in bind-elute mode; the cationexchange chromatography step comprises operating a POROS 50 HSresin-containing column in bind-elute mode, and the HIC step comprisesoperating a PHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin-containingcolumn in flow-through mode.

In still yet another aspect, anti-Abeta monoclonal antibody preparationspurified from CHO cells by a process comprising a hydrophobicinteraction chromatography (HIC) step are provided. In certainembodiments, the purified preparation comprises the anti-Abeta antibodyand a residual amount of hamster PLBL2. In certain embodiments, theamount of hamster PLBL2 is less than 20 ng/mg. In certain embodiments,the amount of hamster PLBL2 is less than 15 ng/mg. In certainembodiments, the amount of hamster PLBL2 is less than 10 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 8 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 5ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than3 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 2 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 1 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 0.5 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15ng/mg, or between 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mg and 8ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3ng/mg, or between 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1ng/mg, or between the limit of assay quantitation (LOQ) and 1 ng/mg. Incertain embodiments, the HIC step comprises PHENYL SEPHAROSE™ 6 FastFlow (High Sub) resin. In certain embodiments, the HIC step comprisesoperating a resin-containing column in flow-through mode. In certainembodiments, the HIC step comprises an equilibration buffer and a washbuffer, wherein each of the equilibration buffer and the wash buffercomprise 150 mM sodium acetate pH 5.0. In certain embodiments, the HICstep comprises an equilibration buffer and a wash buffer, wherein eachof the equilibration buffer and the wash buffer comprise 150 mM sodiumacetate pH 4.0. In certain embodiments, the HIC step comprises anequilibration buffer and a wash buffer, wherein each of theequilibration buffer and the wash buffer comprise 150 mM sodium acetate,240 mM sodium sulfate pH 4.0. In certain embodiments, the HIC stepcomprises an equilibration buffer and a wash buffer, wherein each of theequilibration buffer and the wash buffer comprise 150 mM sodium acetate,240 mM sodium sulfate pH 5.0. In certain embodiments, the load densityis 300 g/L. In certain embodiments, the load density is 100 g/L. Incertain embodiments, the flow-through is monitored by absorbance at 280nanometers and the flow-through is collected between 0.5 OD and for 10column volumes. In certain embodiments, the process further comprises anaffinity chromatography step. In certain embodiments, the affinitychromatography is protein A chromatography. In certain embodiments, theprocess further comprises a mixed mode chromatography step. In certainembodiments, the anti-Abeta antibody comprises three heavy chain CDRs,CDR-H1 having the amino acid sequence of SEQ ID NO.: 23, CDR-H2 havingthe amino acid sequence of SEQ ID NO.: 24, and CDR-H3 having the aminoacid sequence of SEQ ID NO.: 25, and three light chain CDRs, CDR-L1having the amino acid sequence of SEQ ID NO.: 26, CDR-L2 having theamino acid sequence of SEQ ID NO.: 27, and CDR-L3 having the amino acidsequence of SEQ ID NO.: 28. In certain embodiments, the anti-Abetaantibody comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO.: 29. In certain embodiments, the anti-Abetaantibody comprises a light chain variable region having the amino acidsequence of SEQ ID NO.: 30. In certain embodiments, the anti-Abetaantibody comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO.: 29 and a light chain variable region having theamino acid sequence of SEQ ID NO.: 30. In certain embodiments, theamount of hamster PLBL2 is quantified using an immunoassay or a massspectrometry assay. In certain embodiments, the immunoassay is a totalChinese hamster ovary protein ELISA or a hamster PLBL2 ELISA. In certainembodiments, the mass spectrometry assay is LC-MS/MS.

In one aspect, anti-IL17 A/F monoclonal antibody preparations isolatedand purified from CHO cells by a process comprising a hydrophobicinteraction chromatography (HIC) step are provided. In certainembodiments, the purified preparation comprises the anti-IL17 A/Fantibody and a residual amount of hamster PLBL2. In certain embodiments,the amount of hamster PLBL2 is less than 20 ng/mg. In certainembodiments, the amount of hamster PLBL2 is less than 15 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 10 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 8ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than5 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 3 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 2 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 1 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is less than 0.5 ng/mg. In certain embodiments, the amount ofhamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mgand 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mgand 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and3 ng/mg, or between 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1ng/mg, or between the limit of assay quantitation (LOQ) and 1 ng/mg. Incertain embodiments, the HIC step comprises PHENYL SEPHAROSE′ 6 FastFlow (High Sub) resin. In certain embodiments, the HIC step comprisesoperating a resin-containing column in flow-through mode. In certainembodiments, the HIC step comprises an equilibration buffer and a washbuffer, wherein each of the equilibration buffer and the wash buffercomprise 50 mM sodium acetate pH 5.5. In certain embodiments, theflow-through is monitored by absorbance at 280 nanometers and theflow-through is collected between 0.5 OD and for 10 column volumes. Incertain embodiments, the process further comprises an affinitychromatography step. In certain embodiments, the affinity chromatographyis protein A chromatography. In certain embodiments, the process furthercomprises a cation exchange chromatography step. In certain embodiments,the anti-IL17 A/F antibody comprises three heavy chain CDRs, CDR-H1having the amino acid sequence of SEQ ID NO.: 15, CDR-H2 having theamino acid sequence of SEQ ID NO.: 16, and CDR-H3 having the amino acidsequence of SEQ ID NO.: 17, and three light chain CDRs, CDR-L1 havingthe amino acid sequence of SEQ ID NO.: 18, CDR-L2 having the amino acidsequence of SEQ ID NO.: 19, and CDR-L3 having the amino acid sequence ofSEQ ID NO.: 20. In certain embodiments, the anti-IL17 A/F antibodycomprises a heavy chain variable region having the amino acid sequenceof SEQ ID NO.: 21. In certain embodiments, the anti-IL17 A/F antibodycomprises a light chain variable region having the amino acid sequenceof SEQ ID NO.: 22. In certain embodiments, the anti-IL17 A/F antibodycomprises a heavy chain variable region having the amino acid sequenceof SEQ ID NO.: 21 and a light chain variable region having the aminoacid sequence of SEQ ID NO.: 32. In certain embodiments, the amount ofhamster PLBL2 is quantified using an immunoassay or a mass spectrometryassay. In certain embodiments, the immunoassay is a total Chinesehamster ovary protein ELISA or a hamster PLBL2 ELISA. In certainembodiments, the mass spectrometry assay is LC-MS/MS.

In still another aspect, compositions comprising an anti-Abetamonoclonal antibody purified from CHO cells comprising the anti-Abetaantibody and a residual amount of hamster PLBL2 are provided. In certainembodiments, the amount of hamster PLBL2 is less than 20 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 15 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 10ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than8 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 5 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 3 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 2 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is less than 1 ng/mg. In certain embodiments, the amount ofhamster PLBL2 is less than 0.5 ng/mg. In certain embodiments, the amountof hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mgand 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mgand 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and3 ng/mg, or between 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1ng/mg, or between the limit of assay quantitation (LOQ) and 1 ng/mg. Incertain embodiments, the anti-Abeta antibody is crenezumab. In certainembodiments, the anti-Abeta antibody comprises three heavy chain CDRs,CDR-H1 having the amino acid sequence of SEQ ID NO.:23, CDR-H2 havingthe amino acid sequence of SEQ ID NO.:24, and CDR-H3 having the aminoacid sequence of SEQ ID NO.:25, and three light chain CDRs, CDR-L1having the amino acid sequence of SEQ ID NO.:26, CDR-L2 having the aminoacid sequence of SEQ ID NO.:27, and CDR-L3 having the amino acidsequence of SEQ ID NO.:28. In certain embodiments, the anti-Abetaantibody comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO.:29. In certain embodiments, the anti-Abetaantibody comprises a light chain variable region having the amino acidsequence of SEQ ID NO.:30. In certain embodiments, the anti-Abetaantibody comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO.:29 and a light chain variable region having theamino acid sequence of SEQ ID NO.:30.

In yet still another aspect, compositions comprising an anti-IL17 A/Fmonoclonal antibody purified from CHO cells comprising the anti-IL17 A/Fantibody and a residual amount of hamster PLBL2 are provided. In certainembodiments, the composition comprises the anti-IL17 A/F antibody and aresidual amount of hamster PLBL2, wherein the amount of hamster PLBL2 isless than 20 ng/mg, or less than 15 ng/mg, or less than 10 ng/mg, orless than 8 ng/mg, or less than 5 ng/mg, or less than 3 ng/mg, or lessthan 2 ng/mg, or less than 1 ng/mg, or less than 0.5 ng/mg. In certainembodiments, the amount of hamster PLBL2 is less than 20 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 15 ng/mg.In certain embodiments, the amount of hamster PLBL2 is less than 10ng/mg. In certain embodiments, the amount of hamster PLBL2 is less than8 ng/mg. In certain embodiments, the amount of hamster PLBL2 is lessthan 5 ng/mg. In certain embodiments, the amount of hamster PLBL2 isless than 3 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 2 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is less than 1 ng/mg. In certain embodiments, the amount ofhamster PLBL2 is less than 0.5 ng/mg. In certain embodiments, the amountof hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mgand 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg, or between 0.5 ng/mgand 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and3 ng/mg, or between 0.5. ng/mg and 2 ng/mg, or between 0.5 ng/mg and 1ng/mg, or between the limit of assay quantitation (LOQ) and 1 ng/mg. Incertain embodiments, the anti-IL17 A/F antibody comprises three heavychain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.:15,CDR-H2 having the amino acid sequence of SEQ ID NO.:16, and CDR-H3having the amino acid sequence of SEQ ID NO.:17, and three light chainCDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.:18, CDR-L2having the amino acid sequence of SEQ ID NO.:19, and CDR-L3 having theamino acid sequence of SEQ ID NO.:20. In certain embodiments, theanti-IL17 A/F antibody comprises a heavy chain variable region havingthe amino acid sequence of SEQ ID NO.:21. In certain embodiments, theanti-IL17 A/F antibody comprises a light chain variable region havingthe amino acid sequence of SEQ ID NO.:22. In certain embodiments, theanti-IL17 A/F antibody comprises a heavy chain variable region havingthe amino acid sequence of SEQ ID NO.:21 and a light chain variableregion having the amino acid sequence of SEQ ID NO.:22.

In one aspect, methods of treating an IL-13-mediated disorder comprisingadministering a treatment composition comprising an anti-IL13 monoclonalantibody purified from CHO cells and a residual amount of hamster PLBL2are provided. In certain embodiments, the amount of hamster PLBL2 isless than 20 ng/mg. In certain embodiments, the amount of hamster PLBL2is less than 15 ng/mg. In certain embodiments, the amount of hamsterPLBL2 is less than 10 ng/mg. In certain embodiments, the amount ofhamster PLBL2 is less than 8 ng/mg. In certain embodiments, the amountof hamster PLBL2 is less than 5 ng/mg. In certain embodiments, theamount of hamster PLBL2 is less than 3 ng/mg. In certain embodiments,the amount of hamster PLBL2 is less than 2 ng/mg. In certainembodiments, the amount of hamster PLBL2 is less than 1 ng/mg. Incertain embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg.In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng/mgand 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mgand 10 ng/mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5. ng/mg and 2ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of assayquantitation (LOQ) and 1 ng/mg. In certain embodiments, the anti-IL13antibody comprises three heavy chain CDRs, CDR-H1 having the amino acidsequence of SEQ ID NO.: 1, CDR-H2 having the amino acid sequence of SEQID NO.: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO.: 3,and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQID NO.: 4, CDR-L2 having the amino acid sequence of SEQ ID NO.: 5, andCDR-L3 having the amino acid sequence of SEQ ID NO.: 6. In certainembodiments, the anti-IL13 antibody comprises a heavy chain variableregion having the amino acid sequence of SEQ ID NO.: 7. In certainembodiments, the anti-IL13 antibody comprises a light chain variableregion having the amino acid sequence of SEQ ID NO.: 9. In certainembodiments, the anti-IL13 antibody comprises a heavy chain having theamino acid sequence of SEQ ID NO.: 10. In certain embodiments, theanti-IL13 antibody comprises a light chain having the amino acidsequence of SEQ ID NO.: 14. In certain embodiments, the anti-IL13antibody comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO.: 7 and a light chain variable region having theamino acid sequence of SEQ ID NO.: 9. In certain embodiments, theanti-IL13 antibody comprises a heavy chain having the amino acidsequence of SEQ ID NO.: 10 and a light chain having the amino acidsequence of SEQ ID NO.: 14. In certain embodiments, the treatmentcomposition is administered subcutaneously once every four weeks. Incertain embodiments, the treatment composition is administeredsubcutaneously once every eight weeks. In certain embodiments, thetreatment composition is administered subcutaneously once every 12weeks. In certain embodiments, the patient is treated once every fourweeks for at least one month. In certain embodiments, the patient istreated once every four weeks for at least three months. In certainembodiments, the patient is treated once every four weeks for at leastsix months. In certain embodiments, the patient is treated once everyfour weeks for at least nine months. In certain embodiments, the patientis treated once every four weeks for at least 12 months. In certainembodiments, the patient is treated once every four weeks for at least18 months. In certain embodiments, the patient is treated once everyfour weeks for at least two years. In certain embodiments, the patientis treated once every four weeks for more than two years. In certainembodiments, the IL-13-mediated disorder is asthma. In certainembodiments, the IL-13-mediated disorder is idiopathic pulmonaryfibrosis. In certain embodiments, the IL-13-mediated disorder is atopicdermatitis. In certain embodiments, the IL-13-mediated disorder isselected from allergic asthma, non-allergic asthma, allergic rhinitis,allergic conjunctivitis, eczema, urticaria, food allergies, chronicobstructive pulmonary disease, ulcerative colitis, RSV infection,uveitis, scleroderma, and osteoporosis.

In another aspect, administration of a treatment composition to apatient according to any of the methods described above is lessimmunogenic for hamster PLBL2 compared to administration of a referencecomposition, wherein the reference composition comprises an anti-IL13monoclonal antibody purified from Chinese hamster ovary host cells and aresidual amount of hamster PLBL2 of greater than 30 ng/mg. In certainembodiments, the amount of hamster PLBL2 in the reference composition isgreater than 50 ng/mg. In certain embodiments, the amount of hamsterPLBL2 in the reference composition is greater than 100 ng/mg. In certainembodiments, the amount of hamster PLBL2 in the reference composition isgreater than 200 ng/mg. In certain embodiments, the amount of hamsterPLBL2 in the reference composition is greater than 300 ng/mg. In certainembodiments, the amount of hamster PLBL2 in the reference composition isbetween 30 ng/mg and 300 ng/mg, or between 30 ng/mg and 200 ng/mg, orbetween 30 ng/mg and 100 ng/mg, or between 30 ng/mg and 50 ng/mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show total CHOP levels in caprylic acid-treated Protein Apools of anti-IL13 MAb as described in Example 2. (FIG. 1A) Caprylicacid precipitation of Protein A pool at pH 4.5; (FIG. 1B) Caprylic acidprecipitation of Protein A pool at pH 5.0. CHOP levels in ng/mg areindicated along the vertical axis; percentage of caprylic acid is shownalong the horizontal axis, each bar represents the value from 2-foldserial dilution.

FIG. 2 shows total CHOP levels in additive-treated HCCF anti-IL13 MAbfollowing Protein A chromatography which was followed by cation exchangechromatography on POROS® 50HS as described in Example 2. Corrected CHOPlevels in ng/ml are shown on the vertical axis; the additive (control,0.6M guanidine, or 0.6M arginine) is indicated on the horizontal axis,each bar represents the value from 2-fold serial dilution as indicated.

FIGS. 3A-3D show total CHOP levels in UFDF pools of anti-IL13 MAbsubjected to different HIC resins under varying salt and pH conditionsas described in Example 2. (FIG. 3A) OCTYL-SEPHAROSE® Fast Flow resin;(FIG. 3B) PHENYL SEPHAROSE™ 6 Fast Flow (low sub) resin; (FIG. 3C)BUTYL-SEPHAROSE® 4 Fast Flow resin; (FIG. 3D) PHENYL SEPHAROSE™ 6 FastFlow (high sub) resin; highest dilution CHOP (in ppm) is shown on thevertical axis and sodium sulfate concentration is shown on thehorizontal axis; pH (5.5, 6.0, 7.0, or 8.0 is indicated by the legend.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provideone skilled in the art with a general guide to many of the terms used inthe present application.

Certain Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with any document incorporatedherein by reference, the definition set forth below shall control.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a protein”or an “antibody” includes a plurality of proteins or antibodies,respectively; reference to “a cell” includes mixtures of cells, and thelike.

The term “detecting” is used herein in the broadest sense to includeboth qualitative and quantitative measurements of a target molecule.Detecting includes identifying the mere presence of the target moleculein a sample as well as determining whether the target molecule ispresent in the sample at detectable levels.

A “sample” refers to a small portion of a larger quantity of material.Generally, testing according to the methods described herein isperformed on a sample. The sample is typically obtained from arecombinant polypeptide preparation obtained, for example, from culturedhost cells. A sample may be obtained from, for example but not limitedto, harvested cell culture fluid, from an in-process pool at a certainstep in a purification process, or from the final purified product.

The term “product” as described herein is the substance to be purifiedby various chromatographic methods; for example, a polypeptide.

The term “polypeptide” or “protein” are used interchangeably herein torefer to polymers of amino acids of any length. The polymer may belinear or branched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. The terms “polypeptide”and “protein” as used herein specifically encompass antibodies.

“Purified” polypeptide (e.g., antibody or immunoadhesin) means that thepolypeptide has been increased in purity, such that it exists in a formthat is more pure than it exists in its natural environment and/or wheninitially synthesized and/or amplified under laboratory conditions.Purity is a relative term and does not necessarily mean absolute purity.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide fused to a “tag polypeptide.” Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (in certain instances,between about 10 and 20 amino acid residues).

“Active” or “activity” for the purposes herein refers to form(s) of apolypeptide which retain a biological and/or an immunological activityof interest, wherein “biological” activity refers to a biologicalfunction (either inhibitory or stimulatory) caused by the polypeptideother than the ability to induce the production of an antibody againstan antigenic epitope possessed by the polypeptide and an “immunological”activity refers to the ability to induce the production of an antibodyagainst an antigenic epitope possessed by the polypeptide.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide, e.g., a cytokine. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a nativepolypeptide. Suitable agonist or antagonist molecules specificallyinclude agonist or antagonist antibodies or antibody fragments,fragments or amino acid sequence variants of native polypeptides, andthe like. Methods for identifying agonists or antagonists of apolypeptide may comprise contacting a polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the polypeptide.

A polypeptide “which binds” an antigen of interest, e.g. atumor-associated polypeptide antigen target, is one that binds theantigen with sufficient affinity such that the polypeptide is useful asan assay reagent, a diagnostic and/or therapeutic agent in targeting asample containing the antigen, a cell or tissue expressing the antigen,and does not significantly cross-react with other polypeptides.

With regard to the binding of a polypeptide to a target molecule, theterm “specific binding” or “specifically binds to” or is “specific for”a particular polypeptide or an epitope on a particular polypeptidetarget means binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. For example, specific binding can bedetermined by competition with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies) formed from at least two intact antibodies, andantibody fragments so long as they exhibit the desired biologicalactivity. The term “immunoglobulin” (Ig) is used interchangeable withantibody herein.

Antibodies are naturally occurring immunoglobulin molecules which havevarying structures, all based upon the immunoglobulin fold. For example,IgG antibodies have two “heavy” chains and two “light” chains that aredisulphide-bonded to form a functional antibody. Each heavy and lightchain itself comprises a “constant” (C) and a “variable” (V) region. TheV regions determine the antigen binding specificity of the antibody,whilst the C regions provide structural support and function innon-antigen-specific interactions with immune effectors. The antigenbinding specificity of an antibody or antigen-binding fragment of anantibody is the ability of an antibody to specifically bind to aparticular antigen.

The antigen binding specificity of an antibody is determined by thestructural characteristics of the V region. The variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

Each V region typically comprises three complementarity determiningregions (“CDRs”, each of which contains a “hypervariable loop”), andfour framework regions. An antibody binding site, the minimal structuralunit required to bind with substantial affinity to a particular desiredantigen, will therefore typically include the three CDRs, and at leastthree, preferably four, framework regions interspersed there between tohold and present the CDRs in the appropriate conformation. Classicalfour chain antibodies have antigen binding sites which are defined byV_(H) and V_(L) domains in cooperation. Certain antibodies, such ascamel and shark antibodies, lack light chains and rely on binding sitesformed by heavy chains only. Single domain engineered immunoglobulinscan be prepared in which the binding sites are formed by heavy chains orlight chains alone, in absence of cooperation between V_(H) and V_(L).

The term “hypervariable region” when used herein refers to certain aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region may comprise amino acid residues from a“complementarity determining region” or “CDR” as discussed above (e.g.,around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in theV_(L), and around about 31-35B (H1), 50-65 (H2) and 95-102 (H3) in theV_(H) (Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (e.g.residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32(H1), 52A-55 (H2) and 96-101 (H3) in the V_(H) (Chothia and Lesk. J.Mol. Biol. 196:901-917 (1987)).

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat.No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); one-armed antibodies, single variable domain antibodies,minibodies, single-chain antibody molecules; multispecific antibodiesformed from antibody fragments (e.g., including but not limited to,Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem(di,tri)-scFv); and Bi-specific T-cell engagers (BiTEs).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constantdomains that correspond to the different classes of antibodies arecalled α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the scFv to form the desired structure for antigen binding. Fora review of scFv see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody that has polyepitopic specificity. Suchmultispecific antibodies include, but are not limited to, an antibodycomprising a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), where the V_(H)V_(L) unit has polyepitopicspecificity, antibodies having two or more V_(L) and V_(H) domains witheach V_(H)V_(L) unit binding to a different epitope, antibodies havingtwo or more single variable domains with each single variable domainbinding to a different epitope, full length antibodies, antibodyfragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies,triabodies, tri-functional antibodies, antibody fragments that have beenlinked covalently or non-covalently. “Polyepitopic specificity” refersto the ability to specifically bind to two or more different epitopes onthe same or different target(s). “Monospecific” refers to the ability tobind only one epitope. According to one embodiment the multispecificantibody is an IgG antibody that binds to each epitope with an affinityof 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001pM, or 0.1 μM to 0.001 pM.

The expression “single domain antibodies” (sdAbs) or “single variabledomain (SVD) antibodies” generally refers to antibodies in which asingle variable domain (V_(H) or V_(L)) can confer antigen binding. Inother words, the single variable domain does not need to interact withanother variable domain in order to recognize the target antigen.Examples of single domain antibodies include those derived from camelids(lamas and camels) and cartilaginous fish (e.g., nurse sharks) and thosederived from recombinant methods from humans and mouse antibodies(Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; TrendBiochem Sci (2001) 26:230-235; Trends Biotechnol (2003):21:484-490; WO2005/035572; WO 03/035694; Febs Lett (1994) 339:285-290; WO00/29004; WO02/051870).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are uncontaminated by other immunoglobulins.The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the methods provided herein maybe made by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable domain antigen-binding sequences derived from anon-human primate (e.g. Old World Monkey, such as baboon, rhesus orcynomolgus monkey) and human constant region sequences (U.S. Pat. No.5,693,780).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence, except for FRsubstitution(s) as noted above. The humanized antibody optionally alsowill comprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

For the purposes herein, an “intact antibody” is one comprising heavyand light variable domains as well as an Fc region. The constant domainsmay be native sequence constant domains (e.g. human native sequenceconstant domains) or amino acid sequence variant thereof. Preferably,the intact antibody has one or more effector functions.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The terms “anti-IL-13 antibody” and “an antibody that binds to IL-13”refer to an antibody that is capable of binding IL-13 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting IL-13. In some embodiments, the extent ofbinding of an anti-IL-13 antibody to an unrelated, non-IL-13 protein isless than about 10% of the binding of the antibody to IL-13 as measured,e.g., by a radioimmunoassay (MA). In certain embodiments, an antibodythat binds to IL-13 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10-8 M or less,e.g. from 10-8 M to 10-13 M, e.g., from 10-9 M to 10-13 M). In certainembodiments, an anti-IL-13 antibody binds to an epitope of IL-13 that isconserved among IL-13 from different species.

“IL-13 mediated disorder” means a disorder associated with excess IL-13levels or activity in which atypical symptoms may manifest due to thelevels or activity of IL-13 locally and/or systemically in the body.Examples of IL-13 mediated disorders include: cancers (e.g.,non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergicrhinitis, asthma, fibrosis, inflammatory bowel disease, Crohn's disease,lung inflammatory disorders (including pulmonary fibrosis such as IPF),COPD, and hepatic fibrosis.

The term “respiratory disorder” includes, but is not limited to, asthma(e.g., allergic and non-allergic asthma (e.g., due to infection, e.g.,with respiratory syncytial virus (RSV), e.g., in younger children));bronchitis (e.g., chronic bronchitis); chronic obstructive pulmonarydisease (COPD) (e.g., emphysema (e.g., cigarette-induced emphysema);conditions involving airway inflammation, eosinophilia, fibrosis andexcess mucus production, e.g., cystic fibrosis, pulmonary fibrosis, andallergic rhinitis. Examples of diseases that can be characterized byairway inflammation, excessive airway secretion, and airway obstructioninclude asthma, chronic bronchitis, bronchiectasis, and cystic fibrosis.

The term “therapeutic agent” refers to any agent that is used to treat adisease. A therapeutic agent may be, for example, a polypeptide(s)(e.g., an antibody, an immunoadhesin or a peptibody), an aptamer or asmall molecule that can bind to a protein or a nucleic acid moleculethat can bind to a nucleic acid molecule encoding a target (i.e.,siRNA), and the like.

A “naked antibody” is an antibody (as herein defined) that is notconjugated to a heterologous molecule, such as a cytotoxic moiety orradiolabel.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term “sequential” as used herein with regard to chromatographyrefers to having a first chromatography followed by a secondchromatography. Additional steps may be included between the firstchromatography and the second chromatography.

The term “continuous” as used herein with regard to chromatographyrefers to having a first chromatography material and a secondchromatography material either directly connected or some othermechanism which allows for continuous flow between the twochromatography materials.

“Impurities” and “contaminants” refer to materials that are differentfrom the desired polypeptide product. Impurities and contaminantsinclude, without limitation: host cell materials, such as CHOP,including single CHOP species; leached Protein A; nucleic acid; avariant, fragment, aggregate or derivative of the desired polypeptide;another polypeptide; endotoxin; viral contaminant; cell culture mediacomponent, etc. In some examples, the contaminant may be a host cellprotein (HCP) from, for example but not limited to, a bacterial cellsuch as an E. coli cell, an insect cell, a prokaryotic cell, aeukaryotic cell, a yeast cell, a mammalian cell, an avian cell, a fungalcell.

The terms “Chinese hamster ovary cell protein” and “CHOP” are usedinterchangeably to refer to a mixture of host cell proteins (“HCP”)derived from a Chinese hamster ovary (“CHO”) cell culture. The HCP orCHOP is generally present as an impurity in a cell culture medium orlysate (e.g., a harvested cell culture fluid (“HCCF”)) comprising aprotein of interest such as an antibody or immunoadhesin expressed in aCHO cell.) The amount of CHOP present in a mixture comprising a proteinof interest provides a measure of the degree of purity for the proteinof interest. HCP or CHOP includes, but is not limited to, a protein ofinterest expressed by the host cell, such as a CHO host cell. Typically,the amount of CHOP in a protein mixture is expressed in parts permillion relative to the amount of the protein of interest in themixture. It is understood that where the host cell is another mammaliancell type, an E. coli, a yeast, an insect cell, or a plant cell, HCPrefers to the proteins, other than target protein, found in a lysate ofthe host cell.

The term “parts per million” or “ppm” are used interchangeably herein torefer to a measure of purity of the protein of interest purified by amethod of the invention. The units ppm refer to the amount of HCP orCHOP in nanograms/milliliter per protein of interest inmilligrams/milliliter (i.e., CHOP ppm=(CHOP ng/ml)/(protein of interestmg/ml), where the proteins are in solution). Where the proteins aredried (such as by lyophilization), ppm refers to (CHOP ng)/(protein ofinterest mg)). Impurities may also be expressed as “ng/mg” which is usedinterchangeably with ppm.

By “purifying” a polypeptide from a composition comprising thepolypeptide and one or more impurities is meant increasing the degree ofpurity of the polypeptide in the composition by removing (completely orpartially) at least one impurity from the composition.

A “purification step” may be part of an overall purification processresulting in a “homogeneous” composition, which is used herein to referto a composition comprising less than 100 ppm HCP (100 ng/mg) in acomposition comprising the protein of interest, or less than 90 ppm (90ng/mg), or less than 80 ppm (80 ng/mg), or less than 70 ppm (70 ng/mg),or less than 60 ppm (60 ng/mg), or less than 50 ppm 50 ng/mg), or lessthan 40 ppm (40 ng/mg), or less than 30 ppm (30 ng/mg), or less than 20ppm (20 ng/mg), or less than 10 ppm (10 ng/mg), or less than 5 ppm (5ng/mg), or less than 3 ppm (3 ng/mg) or less than 1 ppm (1 ng/mg). Incertain embodiments, the HCP is a single HCP species. In one embodiment,the single HCP species is hamster PLBL2.

The “composition” to be purified herein comprises the polypeptide ofinterest and one or more impurities or contaminants. The composition maybe “partially purified” (i.e. having been subjected to one or morepurification steps or may be obtained directly from a host cell ororganism producing the polypeptide (e.g. the composition may compriseharvested cell culture fluid).

The terms “Protein A” and “ProA” are used interchangeably herein andencompasses Protein A recovered from a native source thereof, Protein Aproduced synthetically (e.g. by peptide synthesis or by recombinanttechniques), and variants thereof which retain the ability to bindproteins which have a CH2/CH3 region, such as an Fc region. Protein Acan be purchased commercially from various sources. Protein A isgenerally immobilized on a solid phase support material. The term “ProA”also refers to an affinity chromatography resin or column containingchromatographic solid support matrix to which is covalently attachedProtein A.

The term “chromatography” refers to the process by which a solute ofinterest in a mixture is separated from other solutes in a mixture as aresult of differences in rates at which the individual solutes of themixture migrate through a stationary medium under the influence of amoving phase, or in bind and elute processes.

The term “affinity chromatography” and “protein affinity chromatography”are used interchangeably herein and refer to a protein separationtechnique in which a protein of interest or antibody of interest isreversibly and specifically bound to a biospecific ligand. Typically,the biospecific ligand is covalently attached to a chromatographic solidphase material and is accessible to the protein of interest in solutionas the solution contacts the chromatographic solid phase material. Theprotein of interest (e.g., antibody, enzyme, or receptor protein)retains its specific binding affinity for the biospecific ligand(antigen, substrate, cofactor, or hormone, for example) during thechromatographic steps, while other solutes and/or proteins in themixture do not bind appreciably or specifically to the ligand. Bindingof the protein of interest to the immobilized ligand allowscontaminating proteins or protein impurities to be passed through thechromatographic medium while the protein of interest remainsspecifically bound to the immobilized ligand on the solid phasematerial. The specifically bound protein of interest is then removed inactive form from the immobilized ligand with low pH, high pH, high salt,competing ligand, and the like, and passed through the chromatographiccolumn with the elution buffer, free of the contaminating proteins orprotein impurities that were earlier allowed to pass through the column.Any component can be used as a ligand for purifying its respectivespecific binding protein, e.g. antibody.

The terms “non-affinity chromatography” and “non-affinity purification”refer to a purification process in which affinity chromatography is notutilized. Non-affinity chromatography includes chromatographictechniques that rely on non-specific interactions between a molecule ofinterest (such as a protein, e.g. antibody) and a solid phase matrix.

The term “specific binding” as used herein in the context ofchromatography, such as to describe interactions between a molecule ofinterest and a ligand bound to a solid phase matrix, refers to thegenerally reversible binding of a protein of interest to a ligandthrough the combined effects of spatial complementarity of protein andligand structures at a binding site coupled with electrostatic forces,hydrogen bonding, hydrophobic forces, and/or van der Waals forces at thebinding site. The greater the spatial complementarity and the strongerthe other forces at the binding site, the greater will be the bindingspecificity of a protein for its respective ligand. Non-limitingexamples of specific binding includes antibody-antigen binding,enzyme-substrate binding, enzyme-cofactor binding, metal ion chelation,DNA binding protein-DNA binding, regulatory protein-proteininteractions, and the like. Typically, in affinity chromatographyspecific binding occurs with an affinity of about 10⁻⁴ to 10⁻⁸ M in freesolution.

The term “non-specific binding” as used herein in the context ofchromatography, such as to describe interactions between a molecule ofinterest and a ligand or other compound bound to a solid phase matrix,refers to binding of a protein of interest to the ligand or compound ona solid phase matrix through electrostatic forces, hydrogen bonding,hydrophobic forces, and/or van der Waals forces at an interaction site,but lacking structural complementarity that enhances the effects of thenon-structural forces. Examples of non-specific interactions include,but are not limited to, electrostatic, hydrophobic, and van der Waalsforces as well as hydrogen bonding.

A “salt” is a compound formed by the interaction of an acid and a base.Exemplary salts include, but are not limited to, acetate (e.g. sodiumacetate), citrate (e.g. sodium citrate), chloride (e.g. sodiumchloride), sulphate (e.g. sodium sulphate), or a potassium salt.

As used herein, “solvent” refers to a liquid substance capable ofdissolving or dispersing one or more other substances to provide asolution. Solvents include aqueous and organic solvents, where certainorganic solvents include a non-polar solvent, ethanol, methanol,isopropanol, acetonitrile, hexylene glycol, propylene glycol, and2,2-thiodiglycol.

The term “detergent” refers to ionic and nonionic surfactants such aspolysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polysorbate, such as polysorbate 20 (TWEEN 20®) orpolysorbate 80 (TWEEN 80®).

A “polymer” herein is a molecule formed by covalent linkage of two ormore monomers, where the monomers are not amino acid residues. Examplesof polymers include, but are not limited to, polyethyl glycol,polypropyl glycol, and copolymers (e.g. PLURONICS™, PF68 etc),polyethylene glycol (PEG), e.g. PEG 400 and PEG 8000.

The term “ion-exchange” and “ion-exchange chromatography” refers to thechromatographic process in which a solute of interest (such as aprotein) in a mixture interacts with a charged compound linked (such asby covalent attachment) to a solid phase ion exchange material such thatthe solute of interest interacts non-specifically with the chargedcompound more or less than solute impurities or contaminants in themixture. The contaminating solutes in the mixture elute from a column ofthe ion exchange material faster or slower than the solute of interestor are bound to or excluded from the resin relative to the solute ofinterest. “Ion-exchange chromatography” specifically includes cationexchange, anion exchange, and mixed mode chromatography.

The phrase “ion exchange material” refers to a solid phase that isnegatively charged (i.e. a cation exchange resin) or positively charged(i.e. an anion exchange resin). The charge may be provided by attachingone or more charged ligands to the solid phase, e.g. by covalentlinking. Alternatively, or in addition, the charge may be an inherentproperty of the solid phase (e.g. as is the case for silica, which hasan overall negative charge).

By “solid phase” is meant a non-aqueous matrix to which one or morecharged ligands can adhere. The solid phase may be a purificationcolumn, a discontinuous phase of discrete particles, a membrane, orfilter etc. Examples of materials for forming the solid phase includepolysaccharides (such as agarose and cellulose); and other mechanicallystable matrices such as silica (e.g. controlled pore glass),poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles andderivatives of any of the above.

A “cation exchange resin” refers to a solid phase which is negativelycharged, and which thus has free cations for exchange with cations in anaqueous solution passed over or through the solid phase. A negativelycharged ligand attached to the solid phase to form the cation exchangeresin may, e.g., be a carboxylate or sulfonate. Commercially availablecation exchange resins include, but are not limited to,carboxy-methyl-cellulose, sulphopropyl (SP) immobilized on agarose (e.g.SP-SEPHAROSE FAST FLOW (or SP-SEPHAROSE HIGH PERFORMANCE) and sulphonylimmobilized on agarose (e.g. S-SEPHAROSE FAST FLOW), and POROS®HS.

A “mixed mode ion exchange resin” refers to a solid phase which iscovalently modified with cationic, anionic, and hydrophobic moieties.Mixed mode ion exchange is also referred to as “multimodal ionexchange.” Commercially available mixed mode ion exchange resin areavailable, e.g., BAKERBOND ABX containing weak cation exchange groups, alow concentration of anion exchange groups, and hydrophobic ligandsattached to a silica gel solid phase support matrix. Additionalexemplary mixed mode ion exchange resins include, but are not limitedto, CAPTO™ Adhere resin, QMA resin, CAPTO™ MMC resin, MEP HyperCelresin, HEA HyperCel resin, PPA HyperCel resin, or ChromaSorb membrane orSartobind STIC. In some embodiments, the mixed mode material is CAPTO™Adhere resin.

The term “anion exchange resin” is used herein to refer to a solid phasewhich is positively charged, e.g. having one or more positively chargedligands, such as quaternary amino groups, attached thereto. Commerciallyavailable anion exchange resins include DEAE cellulose, QAE SEPHADEX andFAST Q SEPHAROSE™ and Q SEPHAROSE™ FAST FLOW.

A “buffer” is a solution that resists changes in pH by the action of itsacid-base conjugate components. Various buffers which can be employeddepending, for example, on the desired pH of the buffer are described inBuffers. A Guide for the Preparation and Use of Buffers in BiologicalSystems, Gueffroy, D., ed. Calbiochem Corporation (1975). In certaininstances, the buffer has a pH in the range from about 2 to about 9,alternatively from about 3 to about 8, alternatively from about 4 toabout 7 alternatively from about 5 to about 7. Non-limiting examples ofbuffers that will control the pH in this range include MES, MOPS, MOPSO,Tris, HEPES, phosphate, acetate, citrate, succinate, and ammoniumbuffers, as well as combinations of these.

The term “hydrophobic interaction chromatography” or “HIC” is usedherein to refer to a chromatographic process that separates moleculebased on their hydrophobicity. Exemplary resins that can be used for HICinclude, but are not limited to phenyl-, butyl-, octyl-SEPHAROSE,BUTYL-SEPHAROSE® 4 Fast Flow, PHENYL SEPHAROSE™ High Performance, PHENYLSEPHAROSE™ 6 Fast Flow (low sub), and PHENYL SEPHAROSE™ 6 Fast Flow(high sub). Typically, sample molecules in a high salt buffer are loadedonto the HIC column. The salt in the buffer interacts with watermolecules to reduce the solvation of the molecules in solution, therebyexposing hydrophobic regions in the sample molecules which areconsequently adsorbed by the HIC column. The more hydrophobic themolecule, the less salt needed to promote binding. Typically, adecreasing salt gradient is used to elute samples from the column. Asthe ionic strength decreases, the exposure of the hydrophilic regions ofthe molecules increases and molecules elute from the column in order ofincreasing hydrophobicity. Sample elution may also be achieved by theaddition of mild organic modifiers or detergents to the elution buffer.

The “loading buffer” is that which is used to load the compositioncomprising the polypeptide molecule of interest and one or moreimpurities onto the ion exchange resin. The loading buffer has aconductivity and/or pH such that the polypeptide molecule of interest(and generally one or more impurities) is/are bound to the ion exchangeresin or such that the protein of interest flows through the columnwhile the impurities bind to the resin.

The “intermediate buffer” is used to elute one or more impurities fromthe ion exchange resin, prior to eluting the polypeptide molecule ofinterest. The conductivity and/or pH of the intermediate buffer is/aresuch that one or more impurity is eluted from the ion exchange resin,but not significant amounts of the polypeptide of interest.

The term “wash buffer” when used herein refers to a buffer used to washor re-equilibrate the ion exchange resin, prior to eluting thepolypeptide molecule of interest. In certain instances, for convenience,the wash buffer and loading buffer may be the same, but this is notrequired.

The “elution buffer” is used to elute the polypeptide of interest fromthe solid phase. The conductivity and/or pH of the elution buffer is/aresuch that the polypeptide of interest is eluted from the ion exchangeresin.

A “regeneration buffer” may be used to regenerate the ion exchange resinsuch that it can be re-used. The regeneration buffer has a conductivityand/or pH as required to remove substantially all impurities and thepolypeptide of interest from the ion exchange resin.

The term “conductivity” refers to the ability of an aqueous solution toconduct an electric current between two electrodes. In solution, thecurrent flows by ion transport. Therefore, with an increasing amount ofions present in the aqueous solution, the solution will have a higherconductivity. The unit of measurement for conductivity is milliSeimensper centimeter (mS/cm), and can be measured using a conductivity metersold, e.g., by Orion. The conductivity of a solution may be altered bychanging the concentration of ions therein. For example, theconcentration of a buffering agent and/or concentration of a salt (e.g.NaCl or KCl) in the solution may be altered in order to achieve thedesired conductivity.

The “pI” or “isoelectric point” of a polypeptide refer to the pH atwhich the polypeptide's positive charge balances its negative charge. pIcan be calculated from the net charge of the amino acid residues orsialic acid residues of attached carbohydrates of the polypeptide or canbe determined by isoelectric focusing.

By “binding” a molecule to an ion exchange material is meant exposingthe molecule to the ion exchange material under appropriate conditions(pH/conductivity) such that the molecule is reversibly immobilized in oron the ion exchange material by virtue of ionic interactions between themolecule and a charged group or charged groups of the ion exchangematerial.

By “washing” the ion exchange material is meant passing an appropriatebuffer through or over the ion exchange material.

To “elute” a molecule (e.g. polypeptide or impurity) from an ionexchange material is meant to remove the molecule therefrom by alteringthe ionic strength of the buffer surrounding the ion exchange materialsuch that the buffer competes with the molecule for the charged sites onthe ion exchange material.

“Ultrafiltration” is a form of membrane filtration in which hydrostaticpressure forces a liquid against a semipermeable membrane. Suspendedsolids and solutes of high molecular weight are retained, while waterand low molecular weight solutes pass through the membrane. In someexamples, ultrafiltration membranes have pore sizes in the range of 1 to100 nm. The terms “ultrafiltration membrane” and “ultrafiltrationfilter” may be used interchangeably.

“Diafiltration” is a method that incorporates ultrafiltration membranesto remove salts or other microsolutes from a solution. Small moleculesare separated from a solution while retaining larger molecules in theretentate. The process selectively utilizes permeable (porous) membranefilters to separate the components of solutions and suspensions based ontheir molecular size.

As used herein, “filtrate” refers to that portion of a sample thatpasses through the filtration membrane.

As used herein, “retentate” refers to that portion of a sample that issubstantially retained by the filtration membrane.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies areused to delay development of a disease or to slow the progression of adisease.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

Anti-IL13 Antibodies

In some embodiments, isolated and purified antibodies that bind IL-13are provided. Exemplary anti-IL13 antibodies are known and include, forexample, but not limited to, lebrikizumab, IMA-026, IMA-638 (alsoreferred to as, anrukinzumab, INN No. 910649-32-0; QAX-576),tralokinumab (also referred to as CAT-354, CAS No. 1044515-88-9);AER-001, ABT-308 (also referred to as humanized 13C5.5 antibody.Examples of such anti-IL13 antibodies and other inhibitors of IL13 aredisclosed, for example, in WO 2005/062967, WO2008/086395, WO2006/085938,U.S. Pat. No. 7,615,213, U.S. Pat. No. 7,501,121, WO2007/036745,WO2010/073119, WO2007/045477. In one embodiment, the anti-IL13 antibodyis a humanized IgG4 antibody. In one embodiment, the anti-IL13 antibodyis lebrikizumab. In one embodiment, the anti-IL13 antibody comprisesthree heavy chain CDRs, CDR-H1 (SEQ ID NO.: 1), CDR-H2 (SEQ ID NO.: 2),and CDR-H3 (SEQ ID NO.: 3). In one embodiment, the anti-IL13 antibodycomprises three light chain CDRS, CDR-L1 (SEQ ID NO.: 4), CDR-L2 (SEQ IDNO.: 5), and CDR-L3 (SEQ ID NO.: 6). In one embodiment, the anti-IL13antibody comprises three heavy chain CDRs and three light chain CDRs,CDR-H1 (SEQ ID NO.: 1), CDR-H2 (SEQ ID NO.: 2), CDR-H3 (SEQ ID NO.: 3),CDR-L1 (SEQ ID NO.: 4), CDR-L2 (SEQ ID NO.: 5), and CDR-L3 (SEQ ID NO.:6). In one embodiment, the anti-IL13 antibody comprises a variable heavychain region, V_(H), having an amino acid sequence selected from SEQ IDNOs. 7 and 8. In one embodiment, the anti-IL13 antibody comprises avariable light chain region, VL, having the amino acid sequence of SEQID NO.: 9. In one embodiment, the anti-IL13 antibody comprises avariable heavy chain region, VH, having an amino acid sequence selectedfrom SEQ ID NOs. 7 and 8 and a variable light chain region, VL, havingan amino acid sequence of SEQ ID NO.: 9. In one embodiment, theanti-IL13 antibody comprises a heavy chain having the amino acidsequence of SEQ ID NO.: 10 or SEQ ID NO.: 11 or SEQ ID NO.: 12 or SEQ IDNO.: 13. In one embodiment, the anti-IL13 antibody comprises a lightchain having the amino acid sequence of SEQ ID NO.: 14. In oneembodiment, the anti-IL13 antibody comprises a heavy chain having anamino acid sequence selected from SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ IDNO.: 12, and SEQ ID NO.: 13 and a light chain having the amino acidsequence of SEQ ID NO.: 14.

In another aspect, an anti-IL-13 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO.: 8. In certain embodiments, a V_(H) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-IL-13 antibody comprising that sequence retains the ability to bindto human IL-13. In certain embodiments, a total of 1 to 10 amino acidshave been substituted, altered inserted and/or deleted in SEQ ID NO.: 8.In certain embodiments, substitutions, insertions, or deletions occur inregions outside the CDRs (i.e., in the FRs). Optionally, the anti-IL13antibody comprises the VH sequence in SEQ ID NO.: 8, includingpost-translational modifications of that sequence.

In another aspect, an anti-IL-13 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO.: 9. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-IL-13 antibody comprising that sequenceretains the ability to bind to IL-13. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO.: 9. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the CDRs (i.e., in the FRs).Optionally, the anti-IL-13 antibody comprises the VL sequence in SEQ IDNO.: 9, including post-translational modifications of that sequence.

In yet another embodiment, the anti-IL-13 antibody comprises a VL regionhaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO.: 9 and aVH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO.:8.

The table below shows the amino acid sequences of the CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions of lebrikizumab, along withVH, VL, heavy chain sequences and light chain sequences. As indicated inTable 1 below, VH and the heavy chain may include an N-terminalglutamine and the heavy chain may also include a C-terminal lysine. Asis well known in the art, N-terminal glutamine residues can formpyroglutamate and C-terminal lysine residues can be clipped duringmanufacturing processes.

TABLE 1 Anti-IL13 antibody (lebrikizumab) amino acid sequences. CDR-H1Ala Tyr Ser Val Asn (SEQ ID NO.: 1) CDR-H2Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys (SEQ ID SerNO.: 2) CDR-H3 Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn (SEQ ID NO.: 3)CDR-L1 Arg Ala Ser Lys Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His(SEQ ID NO.: 4) CDR-L2 Leu Ala Ser Asn Leu Glu Ser (SEQ ID NO.: 5)CDR-L3 Gln Gln Asn Asn Glu Asp Pro Arg Thr (SEQ ID NO.: 6) VHVal Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr (SEQ IDLeu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ser NO.: 7)Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu AlaMet Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys SerArg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu ThrMet Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala GlyAsp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser LeuVal Thr Val Ser Ser VHGln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln (SEQ IDThr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr NO.: 8)Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp LeuAla Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu LysSer Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val LeuThr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys AlaGly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly SerLeu Val Thr Val Ser Ser VLAsp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly (SEQ IDGlu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Asp Ser Tyr NO.: 9)Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro ProLys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn AsnGlu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg H ChainVTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM (SEQ IDIWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY NO. 10)YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDYFPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYTCNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLMISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRVVSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLPPSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDGSFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLG H ChainQVTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM (SEQ IDIWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY NO.: 11)YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDYFPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYTCNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLMISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRVVSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLPPSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDGSFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLG H ChainVTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM (SEQ IDIWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY NO.: 12)YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDYFPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYTCNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLMISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRVVSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLPPSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDGSFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK H ChainQVTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM (SEQ IDIWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY NO.: 13)YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDYFPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYTCNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLMISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRVVSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLPPSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDGSFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK L ChainDIVMTQSPDS LSVSLGERAT INCRASKSVD SYGNSFMHWY QQKPGQPPKL (SEQ IDLIYLASNLES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQQNNEDPR NO.. 14)TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC

Other Recombinant Polypeptides

Recombinant polypeptides produced in CHO cells may be purified accordingto the methods described herein to remove or reduce levels of hamsterPLBL2 such that only residual amounts or an undetectable amount remain.Such polypeptides include, without limitation, growth factors,cytokines, immunoglobulins, antibodies, peptibodies and the like.

Certain exemplary antibodies include antibodies to Abeta, antibodies toIL17A/F and antibodies to CMV. Exemplary anti-Abeta antibodies andmethods of producing such antibodies have been described previously, forexample, in WO2008011348, WO2007068429, WO2001062801, and WO2004071408.Exemplary anti-IL17 A/F antibodies and methods of producing suchantibodies have been described previously, for example, in WO 2009136286and U.S. Pat. No. 8,715,669. Exemplary anti-CMV antibodies, includinganti-CMV-MSL, and methods of producing such antibodies have beendescribed previously, for example, in WO 2012047732.

Exemplary polypeptides include include mammalian proteins, such as,e.g., CD4, integrins and their subunits, such as beta7, growth hormone,including human growth hormone and bovine growth hormone; growth hormonereleasing factor; parathyroid hormone; thyroid stimulating hormone;lipoproteins; ct-1-antitrypsin; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; clotting factors such as factor VIIIC, factor IX,tissue factor, and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or tissue-type plasminogen activator (t-PA,e.g., Activase®, TNKase®, Retevase®); bombazine; thrombin; tumornecrosis factor-α and -β; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-I-a); serum albumin such as human serum albumin;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;DNase; inhibin; activin; vascular endothelial growth factor (VEGF); IgE,receptors for hormones or growth factors; an integrin; protein A or D;rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-α and TGF-β including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I); insulin-like growth factor binding proteins; other CDproteins such as CD3, CD8, CD19 and CD20; erythropoietin (EPO);thrombopoietin (TPO); osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-α, -β, or-γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;interleukins (ILs), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33 and so on; superoxidedismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor (DAF); a viral antigen such as, for example, aportion of an HIV envelope; transport proteins; homing receptors;addressins; regulatory proteins; integrins such as CDlla, CDllb, CDllc,CD18, integrin subunits such alpha4, alphaE, beta7; cellular adhesionmolecules such as an ICAM, VLA-4 and VCAM; a tumor associated antigensuch as HER1, (EGFR), HER2, HER3 or HER4 receptor; Apo2L/TRAIL, andfragments of any of the above listed polypeptides; as well asimmunoadhesins and antibodies binding to; and biologically activefragments or variants of any of the above-listed proteins.

Additional exemplary polypeptides include brain polypeptides, includingbut not limited to, beta-secretase 1 (BACE1), Abeta, epidermal growthfactor receptor (EGFR), human epidermal growth factor receptor 2 (HER2),tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), P-selectin,and caspase 6, and fragments of any of the above listed polypeptides; aswell as immunoadhesins and antibodies binding to; and biologicallyactive fragments or variants of any of the above-listed proteins.

Further exemplary polypeptides include therapeutic antibodies andimmunoadhesins, including, without limitation, antibodies, includingantibody fragments, to one or more of the following antigens: HER1(EGFR), HER2 (e.g., trastuzumab, pertuzumab), HER3, HER4, VEGF (e.g.,bevacizumab, ranibizumab), MET (e.g., onartuzumab), CD20 (e.g.,rituximab, obinutuzumab, ocrelizumab), CD22, CD11a, CD11b, CD11c, CD18,an ICAM, VLA-4, VCAM, IL-17A and/or F, IgE (e.g., omalizumab), DRS,CD40, Apo2L/TRAIL, EGFL7 (e.g., parsatuzumab), NRP1, integrin beta7(e.g., etrolizumab), IL-13 (e.g., lebrikizumab), Abeta (e.g.,crenezumab, gantenerumab), P-selectin (e.g., inclacumab), IL-6R (e.g.,tociluzumab), IFNα (e.g., rontalizumab), Mlprime (e.g., quilizumab),mitogen activated protein kinase (MAPK), OX40L, TSLP, Factor D (e.g.,lampalizumab) and receptors such as: IL-9 receptor, IL-5 receptor,IL-4receptor alpha, IL-13receptoralpha1 and IL-13receptoralpha2, OX40,TSLP-R, IL-7Ralpha (a co-receptor for TSLP), IL17RB (receptor forIL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2, FcepsilonRI andFcepsilonRII/CD23 (receptors for IgE). Other exemplary antibodiesinclude those selected from, and without limitation, antiestrogenreceptor antibody, anti-progesterone receptor antibody, anti-p53antibody, anticathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherinantibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEAantibody, anti-retinoblastoma protein antibody, anti-ras oncoproteinantibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNAantibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody,anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD10antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody,anti-CD15 antibody, anti-CD19 antibody, anti-CD23 antibody, anti-CD30antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody,anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody,anti-LCA/CD45 antibody, anti-CD45RO antibody, anti-CD45RA antibody,anti-CD39 antibody, anti-CD100 antibody, anti-CD95/Fas antibody,anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody,anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratins antibody,anti-vimentins antibody, anti-HPV proteins antibody, anti-kappa lightchains antibody, anti-lambda light chains antibody, anti-melanosomesantibody, anti-prostate specific antigen antibody, anti-S-100 antibody,anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibodyand anti-Tn-antigen antibody.

Certain Purification Methods

The protein to be purified using the methods described herein isgenerally produced using recombinant techniques. Methods for producingrecombinant proteins are described, e.g., in U.S. Pat. Nos. 5,534,615and 4,816,567, specifically incorporated herein by reference. In certainembodiments, the protein of interest is produced in a CHO cell (see,e.g. WO 94/11026). Examples of proteins, including anti-IL13 monoclonalantibodies (anti-IL13 MAb), which can be purified using the processesdescribed herein have been described above.

When using recombinant techniques, the protein can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the protein is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, is removed,for example, by centrifugation or ultrafiltration. Where the protein issecreted into the medium, the recombinant host cells may be separatedfrom the cell culture medium by tangential flow filtration, for example.

Protein A immobilized on a solid phase is used to purify the anti-IL13MAb preparation. In certain embodiments, the solid phase is a columncomprising a glass, silica, agarose or polystyrene surface forimmobilizing the Protein A. In certain embodiments, the solid phase is acontrolled pore glass column or a silicic acid column. Sometimes, thecolumn has been coated with a reagent, such as glycerol, in an attemptto prevent nonspecific adherence to the column. The PROSEP A™ column,commercially available from Bioprocessing Limited, is an example of aProtein A controlled pore glass column which is coated with glycerol.Other examples of columns contemplated herein include the POROS® 50 ATM(polystyrene) column or rProtein A SEPHAROSE FAST FLOW™ (agarose) columnor MABSELECT SURE™ (agarose) column available from GE Healthcare LifeSciences (agarose).

The solid phase for the Protein A chromatography is equilibrated with asuitable buffer. For example, the equilibration buffer may be 25 mMTris, 25 mM NaCl, pH 7.70±0.20.

The preparation derived from the recombinant host cells and containingimpurities and/or contaminants is loaded on the equilibrated solid phaseusing a loading buffer which may be the same as the equilibrationbuffer. As the preparation containing impurities/contaminants flowsthrough the solid phase, the protein is adsorbed to the immobilizedProtein A and other impurities/contaminants (such as Chinese HamsterOvary Proteins, CHOP, where the protein is produced in a CHO cell) maybind nonspecifically to the solid phase.

The next step performed sequentially entails removing theimpurities/contaminants bound to the solid phase, antibody and/orProtein A, by washing the solid phase in an intermediate wash step.After loading, the solid phase may be equilibrated with equilibrationbuffer before beginning the intermediate wash step.

The intermediate wash buffer may comprise salt and optionally a furthercompound, such as (a) detergent (for example, polysorbate, e.g.polysorbate 20 or polysorbate 80); (b) solvent (such as hexyleneglycol); and (c) polymer (such as polyethylene glycol {PEG]).

The salt employed may be selected based on the protein of interest.Exemplary salts include, but are not limited to, sodium acetate, sodiumcitrate, and potassium phosphate.

The amounts of the salt and further compound (if any) in the compositionare such that the combined amount elutes theimpurity(ies)/contaminant(s), without substantially removing the proteinof interest. Exemplary salt concentrations in such wash buffers are fromabout 0.1 to about 2M, or from about 0.2M to about 0.6M. Usefuldetergent concentrations are from about 0.01 to about 5%, or from about0.1 to 1%, or about 0.5%, e.g. where the detergent is polysorbate.Exemplary solvent concentrations are from about 1% to 40%, or from about5 to about 25%. Where the further compound is a polymer (e.g. PEG 400 orPEG 8000), the concentration thereof may, for example, be from about 1%to about 20%, or from about 5% to about 15%.

The pH of the intermediate wash buffer is typically from about 4 toabout 8, or from about 4.5 to about 5.5, or about 5.0. In oneembodiment, the pH is 7.00±0.10.

Following the intermediate wash step described above, the protein ofinterest is recovered from the column. This is typically achieved usinga suitable elution buffer. The protein may, for example, be eluted fromthe column using an elution buffer having a low pH (also referred to asacidic conditions), e.g. in the range from about 2 to about 5, or in therange from about 2.5 to about 3.5. Examples of elution buffers for thispurpose include citrate or acetate buffers.

The eluted protein preparation may be subjected to additionalpurification steps either prior to, or after, the Protein Achromatography step. Exemplary further purification steps includehydroxyapatite chromatography; dialysis; affinity chromatography usingan antibody to capture the protein; hydrophobic interactionchromatography (HIC); ammonium sulphate precipitation; anion or cationexchange chromatography; ethanol precipitation; reverse phase HPLC;chromatography on silica; chromatofocusing;ultrafiltration-diafiltration (UFDF), and gel filtration. In theexamples herein, the Protein A chromatography step is followed bydownstream anion exchange (e.g., Q-Sepharose-Fast Flow) or multimodal(e.g. mixed-mode) ion exchange (e.g., CAPTO™ Adhere) and HIC (e.g.,PHENYL SEPHAROSE™ 6 fast flow-high sub) purification steps.

The protein thus recovered may be formulated in a pharmaceuticallyacceptable carrier and is used for various diagnostic, therapeutic orother uses known for such molecules.

In some embodiments of any of the methods described herein, thechromatography material is an ion exchange chromatography material; forexample, an anion exchange chromatography material. In some embodiments,the anion exchange chromatography material is a solid phase that ispositively charged and has free anions for exchange with anions in anaqueous solution passed over or through the solid phase. In someembodiments of any of the methods described herein, the anion exchangematerial may be a membrane, a monolith, or resin. In an embodiment, theanion exchange material may be a resin. In some embodiments, the anionexchange material may comprise a primary amine, a secondary amine, atertiary amine or a quarternary ammonium ion functional group, apolyamine functional group, or a diethylaminoaethyl functional group. Insome embodiments of the above, the anion exchange chromatographymaterial is an anion exchange chromatography column. In some embodimentsof the above, the anion exchange chromatography material is an anionexchange chromatography membrane.

In some embodiments of any of the methods described herein, the ionexchange material may utilize a conventional chromatography material ora convective chromatography material. The conventional chromatographymaterials include, for example, perfusive materials (e.g.,poly(styrene-divinylbenzene) resin) and diffusive materials (e.g.,cross-linked agarose resin). In some embodiments, thepoly(styrene-divinylbenzene) resin can be POROS® resin. In someembodiments, the cross-linked agarose resin may besulphopropyl-Sepharose Fast Flow (“SPSFF”) resin. The convectivechromatography material may be a membrane (e.g., polyethersulfone) ormonolith material (e.g. cross-linked polymer). The polyethersulfonemembrane may be Mustang. The cross-linked polymer monolith material maybe cross-linked poly(glycidyl methacrylate-co-ethylene dimethacrylate).

Examples of anion exchange materials include, but are not limited to,POROS® HQ 50, POROS® PI 50, POROS® D, Mustang Q, Q SEPHAROSE™ FF, andDEAE Sepharose.

In some aspects, the chromatography material is a hydrophobicinteraction chromatography material. Hydrophobic interactionchromatography (HIC) is a liquid chromatography technique that separatesbiomolecules according to hydrophobicity. Examples of HIC chromatographymaterials include, but are not limited to, Toyopearl hexyl 650,Toyopearl butyl 650, Toyopearl phenyl 650, Toyopearl ether 650, Source,Resource, Sepharose Hi-Trap, Octyl sepharose, PHENYL SEPHAROSE™ highperformance, PHENYL SEPHAROSE™ 6 fast flow (low sub) and PHENYLSEPHAROSE™ 6 fast flow (high sub). In some embodiments of the above, theHIC chromatography material is a HIC chromatography column. In someembodiments of the above, the HIC chromatography material is a HICchromatography membrane.

In some aspects, the chromatography material is an affinitychromatography material. Examples of affinity chromatography materialsinclude, but are not limited to chromatography materials derivatizedwith protein A or protein G. Examples of affinity chromatographymaterial include, but are not limited to, Prosep-VA, Prosep-VA UltraPlus, Protein A sepharose fast flow, Tyopearl Protein A, MAb Select,MABSELECT SURE™ and MABSELECT SURE™ LX. In some embodiments of theabove, the affinity chromatography material is an affinitychromatography column. In some embodiments of the above, the affinitychromatography material is an affinity chromatography membrane.

Various buffers which can be employed depending, for example, on thedesired pH of the buffer, the desired conductivity of the buffer, thecharacteristics of the protein of interest, and the purification method.In some embodiments of any of the methods described herein, the methodscomprise using a buffer. The buffer can be a loading buffer, anequilibration buffer, or a wash buffer. In some embodiments, one or moreof the loading buffer, the equilibration buffer, and/or the wash bufferare the same. In some embodiments, the loading buffer, the equilibrationbuffer, and/or the wash buffer are different. In some embodiments of anyof the methods described herein, the buffer comprises a salt. Theloading buffer may comprise sodium chloride, sodium acetate, or amixture thereof. In some embodiments, the loading buffer is a sodiumchloride buffer. In some embodiments, the loading buffer is a sodiumacetate buffer.

Load, as used herein, is the composition loaded onto a chromatographymaterial. Loading buffer is the buffer used to load the compositioncomprising the product of interest onto a chromatography material. Thechromatography material may be equilibrated with an equilibration bufferprior to loading the composition which is to be purified. In someexamples, the wash buffer is used after loading the composition onto achromatography material and before elution of the polypeptide ofinterest from the solid phase. However, some of the product of interest,e.g. a polypeptide, may be removed from the chromatography material bythe wash buffer (e.g. flow-through mode).

Elution, as used herein, is the removal of the product, e.g.polypeptide, from the chromatography material. Elution buffer is thebuffer used to elute the polypeptide or other product of interest from achromatography material. In many cases, an elution buffer has adifferent physical characteristic than the load buffer. For example, theelution buffer may have a different conductivity than load buffer or adifferent pH than the load buffer. In some embodiments, the elutionbuffer has a lower conductivity than the load buffer. In someembodiments, the elution buffer has a higher conductivity than the loadbuffer. In some embodiments, the elution buffer has a lower pH than theload buffer. In some embodiments, the elution buffer has a higher pHthan the load buffer. In some embodiments the elution buffer has adifferent conductivity and a different pH than the load buffer. Theelution buffer can have any combination of higher or lower conductivityand higher or lower pH.

Conductivity refers to the ability of an aqueous solution to conduct anelectric current between two electrodes. In solution, the current flowsby ion transport. Therefore, with an increasing amount of ions presentin the aqueous solution, the solution will have a higher conductivity.The basic unit of measure for conductivity is the Siemen (or mho), mho(mS/cm), and can be measured using a conductivity meter, such as variousmodels of Orion conductivity meters. Since electrolytic conductivity isthe capacity of ions in a solution to carry electrical current, theconductivity of a solution may be altered by changing the concentrationof ions therein. For example, the concentration of a buffering agentand/or the concentration of a salt (e.g. sodium chloride, sodiumacetate, or potassium chloride) in the solution may be altered in orderto achieve the desired conductivity. Preferably, the salt concentrationof the various buffers is modified to achieve the desired conductivity.

In some embodiments of any of the methods described herein, the flowrate is less than about any of 50 CV/hr, 40 CV/hr, or 30 CV/hr. The flowrate may be between about any of 5 CV/hr and 50 CV/hr, 10 CV/hr and 40CV/hr, or 18 CV/hr and 36 CV/hr. In some embodiments, the flow rate isabout any of 9 CV/hr, 18 CV/hr, 25 CV/hr, 30 CV/hr, 36 CV/hr, or 40CV/hr. In some embodiments of any of the methods described herein, theflow rate is less than about any of 100 cm/hr, 75 cm/hr, or 50 cm/hr.The flow rate may be between about any of 25 cm/hr and 150 cm/hr, 25cm/hr and 100 cm/hr, 50 cm/hr and 100 cm/hr, or 65 cm/hr and 85 cm/hr,or 50 cm/hr and 250 cm/hr, or 100 cm/hr and 250 cm/hr, or 150 cm/hr and250 cm/hr.

Bed height is the height of chromatography material used. In someembodiments of any of the method described herein, the bed height isgreater than about any of 3 cm, 10 cm, or 15 cm. The bed height may bebetween about any of 3 cm and 35 cm, 5 cm and 15 cm, 3 cm and 10 cm, or5 cm and 8 cm. In some embodiments, the bed height is about any of 3 cm,5 cm, 10 cm, or 15 cm. In some embodiments, bed height is determinedbased on the amount of polypeptide or contaminants in the load.

In some embodiments, the chromatography is in a column of vessel with avolume of greater than about 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L,2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 25 L, 50 L, 100 L, 200 L,400 L, or 450 L.

In some embodiments, fractions are collected from the chromatography. Insome embodiments, fractions collected are greater than about 0.01 CV,0.02 CV, 0.03 CV, 0.04 CV, 0.05 CV, 0.06 CV, 0.07 CV, 0.08 CV, 0.09 CV,0.1 CV, 0.2 CV, 0.3 CV, 0.4 CV, 0.5 CV, 0.6 CV, 0.7 CV, 0.8 CV, 0.9 CV,1.0 CV, 2.0 CV, 3.0 CV, 4.0 CV, 5.0, CV. In some embodiments, fractionscontaining the product, e.g. polypeptide, are pooled. In someembodiments, fractions containing the polypeptide from the loadfractions and from the elution fractions are pooled. The amount ofpolypeptide in a fraction can be determined by one skilled in the art;for example, the amount of polypeptide in a fraction can be determinedby UV spectroscopy. In some embodiments, fractions containing detectablepolypeptide fragment are pooled.

In some embodiments of any of the methods described herein, the at leastone impurity or contaminant is any one or more of host cell materials,such as CHOP; leached Protein A; nucleic acid; a variant, fragment,aggregate or derivative of the desired polypeptide; another polypeptide;endotoxin; viral contaminant; cell culture media component, gentamicin,etc. In some examples, the impurity or contaminant may be a host cellprotein (HCP) from, for example but not limited to, a bacterial cellsuch as an E. coli cell, an insect cell, a prokaryotic cell, aeukaryotic cell, a yeast cell, a mammalian cell, an avian cell, a fungalcell.

Host cell proteins (HCP) are proteins from the cells in which thepolypeptide was produced. For example, CHOP are proteins from hostcells, i.e., Chinese Hamster Ovary Proteins. The amount of CHOP may bemeasured by enzyme-linked immunosorbent assay (“ELISA”) or massspectrometry. In some embodiments of any of the methods describedherein, the amount of HCP (e.g. CHOP) is reduced by greater than aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. The amountof HCP may be reduced by between about any of 10% and 99%, 30% and 95%,30% and 99%, 50% and 95%, 50% and 99%, 75% and 99%, or 85% and 99%. Insome embodiments, the amount of HCP is reduced by about any of 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98%. In someembodiments, the reduction is determined by comparing the amount of HCPin the composition recovered from a purification step(s) to the amountof HCP in the composition before the purification step(s).

In some embodiments of any of the methods described herein, the methodsfurther comprise recovering the purified polypeptide. In someembodiments, the purified polypeptide is recovered from any of thepurification steps described herein. The chromatography step may beanion exchange chromatography, HIC, or Protein A chromatography. In someembodiments, the first chromatography step is protein A, followed byanion exchange or multimodal ion exchange, followed by HIC.

In some embodiments, the polypeptide is further purified followingchromatography by viral filtration. Viral filtration is the removal ofviral contaminants in a polypeptide purification feedstream. Examples ofviral filtration include ultrafiltration and microfiltration. In someembodiments the polypeptide is purified using a parvovirus filter.

In some embodiments, the polypeptide is concentrated afterchromatography. Examples of concentration methods are known in the artand include but are not limited to ultrafiltration and diafiltration.

In some embodiments of any of the methods described herein, the methodsfurther comprise combining the purified polypeptide of the methods ofpurification with a pharmaceutically acceptable carrier.

Monoclonal Antibodies

In some embodiments, the antibodies purified according to the methods ofthe invention are monoclonal antibodies. Monoclonal antibodies areobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope except for possible variants that ariseduring production of the monoclonal antibody, such variants generallybeing present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete or polyclonal antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as herein described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the polypeptide used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

In some embodiments, the myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, in some embodiments, the myeloma cell lines aremurine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen. Insome embodiments, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (MA) orenzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, polypeptide A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). In some embodiments, the hybridomacells serve as a source of such DNA. Once isolated, the DNA may beplaced into expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese Hamster Ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin polypeptide, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Review articles on recombinantexpression in bacteria of DNA encoding the antibody include Skerra etal., Curr. Opinion in Immunol. 5:256-262 (1993) and Plückthun, Immunol.Revs., 130:151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature 348:552-554 (1990). Clackson etal., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently joiningto the immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

In some embodiments of any of the methods described herein, the antibodyis IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is anIgG monoclonal antibody.

Humanized Antibodies

In some embodiments, the antibody is a humanized antibody. Methods forhumanizing non-human antibodies have been described in the art. In someembodiments, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some hypervariableregion residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)).Another method uses a particular framework region derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chain variable regions. The same framework may be usedfor several different humanized antibodies (Carter et al., Proc. Natl.Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623(1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, in some embodiments of the methods, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablethat illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human Antibodies

In some embodiments, the antibody is a human antibody. As an alternativeto humanization, human antibodies can be generated. For example, it isnow possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (JO gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); andU.S. Pat. Nos. 5,591,669; 5,589,369; and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat polypeptide gene of a filamentous bacteriophage, such as M13 or fd,and displayed as functional antibody fragments on the surface of thephage particle. Because the filamentous particle contains asingle-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. Thus, the phagemimics some of the properties of the B cell. Phage display can beperformed in a variety of formats; for their review see, e.g., Johnson,Kevin S. and Chiswell, David J., Current Opinion in Structural Biology3:564-571 (1993). Several sources of V-gene segments can be used forphage display. Clackson et al., Nature 352:624-628 (1991) isolated adiverse array of anti-oxazolone antibodies from a small randomcombinatorial library of V genes derived from the spleens of immunizedmice. A repertoire of V genes from unimmunized human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffithet al., EMBO J 12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332 and5,573,905.

Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Antibody Fragments

In some embodiments, the antibody is an antibody fragment. Varioustechniques have been developed for the production of antibody fragments.Traditionally, these fragments were derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1992) and Brennan et al., Science229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)2 fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody,” e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

In some embodiments, fragments of the antibodies described herein areprovided. In some embodiments, the antibody fragment is an antigenbinding fragment. In some embodiments, the antigen binding fragment isselected from the group consisting of a Fab fragment, a Fab′ fragment, aF(ab′)₂ fragment, a scFv, a Fv, and a diabody.

Chimeric Polypeptides

The polypeptide described herein may be modified in a way to formchimeric molecules comprising the polypeptide fused to another,heterologous polypeptide or amino acid sequence. In some embodiments, achimeric molecule comprises a fusion of the polypeptide with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the polypeptide. The presence of suchepitope-tagged forms of the polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the polypeptide to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag.

Other

Another type of covalent modification of the polypeptide compriseslinking the polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol. The polypeptide also may be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,18th edition, Gennaro, A. R., Ed., (1990).

Obtaining Polypeptides

The polypeptides used in the methods of purification described hereinmay be obtained using methods well-known in the art, including therecombination methods. The following sections provide guidance regardingthese methods.

Polynucleotides

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.

Polynucleotides encoding polypeptides may be obtained from any sourceincluding, but not limited to, a cDNA library prepared from tissuebelieved to possess the polypeptide mRNA and to express it at adetectable level. Accordingly, polynucleotides encoding polypeptide canbe conveniently obtained from a cDNA library prepared from human tissue.The polypeptide-encoding gene may also be obtained from a genomiclibrary or by known synthetic procedures (e.g., automated nucleic acidsynthesis).

For example, the polynucleotide may encode an entire immunoglobulinmolecule chain, such as a light chain or a heavy chain. A complete heavychain includes not only a heavy chain variable region (V_(H)) but also aheavy chain constant region (C_(H)), which typically will comprise threeconstant domains: C_(H)1, C_(H)2 and C_(H)3; and a “hinge” region. Insome situations, the presence of a constant region is desirable.

Other polypeptides which may be encoded by the polynucleotide includeantigen-binding antibody fragments such as single domain antibodies(“dAbs”), Fv, scFv, Fab′ and F(ab′)₂ and “minibodies.” Minibodies are(typically) bivalent antibody fragments from which the C_(H)1 and C_(K)or C_(L) domain has been excised. As minibodies are smaller thanconventional antibodies they should achieve better tissue penetration inclinical/diagnostic use, but being bivalent they should retain higherbinding affinity than monovalent antibody fragments, such as dAbs.Accordingly, unless the context dictates otherwise, the term “antibody”as used herein encompasses not only whole antibody molecules but alsoantigen-binding antibody fragments of the type discussed above.Preferably each framework region present in the encoded polypeptide willcomprise at least one amino acid substitution relative to thecorresponding human acceptor framework. Thus, for example, the frameworkregions may comprise, in total, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acidsubstitutions relative to the acceptor framework regions.

Suitably, the polynucleotides described herein may be isolated and/orpurified. In some embodiments, the polynucleotides are isolatedpolynucleotides.

The term “isolated polynucleotide” is intended to indicate that themolecule is removed or separated from its normal or natural environmentor has been produced in such a way that it is not present in its normalor natural environment. In some embodiments, the polynucleotides arepurified polynucleotides. The term purified is intended to indicate thatat least some contaminating molecules or substances have been removed.

Suitably, the polynucleotides are substantially purified, such that therelevant polynucleotides constitutes the dominant (i.e., most abundant)polynucleotides present in a composition.

Expression of Polynucleotides

The description below relates primarily to production of polypeptides byculturing cells transformed or transfected with a vector containingpolypeptide-encoding polynucleotides. It is, of course, contemplatedthat alternative methods, which are well known in the art, may beemployed to prepare polypeptides. For instance, the appropriate aminoacid sequence, or portions thereof, may be produced by direct peptidesynthesis using solid-phase techniques (see, e.g., Stewart et al.,Solid-Phase Peptide Synthesis W.H. Freeman Co., San Francisco, Calif.(1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). In vitroprotein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) usingmanufacturer's instructions. Various portions of the polypeptide may bechemically synthesized separately and combined using chemical orenzymatic methods to produce the desired polypeptide.

Polynucleotides as described herein are inserted into an expressionvector(s) for production of the polypeptides. The term “controlsequences” refers to DNA sequences necessary for the expression of anoperably linked coding sequence in a particular host organism. Thecontrol sequences include, but are not limited to, promoters (e.g.,naturally-associated or heterologous promoters), signal sequences,enhancer elements, and transcription termination sequences.

A polynucleotide is “operably linked” when it is placed into afunctional relationship with another polynucleotide sequence. Forexample, nucleic acids for a presequence or secretory leader is operablylinked to nucleic acids for a polypeptide if it is expressed as apreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that thenucleic acid sequences being linked are contiguous, and, in the case ofa secretory leader, contiguous and in reading phase. However, enhancersdo not have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

For antibodies, the light and heavy chains can be cloned in the same ordifferent expression vectors. The nucleic acid segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides.

The vectors containing the polynucleotide sequences (e.g., the variableheavy and/or variable light chain encoding sequences and optionalexpression control sequences) can be transferred into a host cell bywell-known methods, which vary depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics or viral-based transfection may be used forother cellular hosts. (See generally Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). Othermethods used to transform mammalian cells include the use of polybrene,protoplast fusion, liposomes, electroporation, and microinjection. Forproduction of transgenic animals, transgenes can be microinjected intofertilized oocytes, or can be incorporated into the genome of embryonicstem cells, and the nuclei of such cells transferred into enucleatedoocytes.

Vectors

The term “vector” includes expression vectors and transformation vectorsand shuttle vectors.

The term “expression vector” means a construct capable of in vivo or invitro expression.

The term “transformation vector” means a construct capable of beingtransferred from one entity to another entity—which may be of thespecies or may be of a different species. If the construct is capable ofbeing transferred from one species to another—such as from anEscherichia coli plasmid to a bacterium, such as of the genus Bacillus,then the transformation vector is sometimes called a “shuttle vector”.It may even be a construct capable of being transferred from an E. coliplasmid to an Agrobacterium to a plant.

Vectors may be transformed into a suitable host cell as described belowto provide for expression of a polypeptide. Various vectors are publiclyavailable. The vector may, for example, be in the form of a plasmid,cosmid, viral particle, or phage. The appropriate nucleic acid sequencemay be inserted into the vector by a variety of procedures. In general,DNA is inserted into an appropriate restriction endonuclease site(s)using techniques known in the art. Construction of suitable vectorscontaining one or more of these components employs standard ligationtechniques which are known to the skilled artisan.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.Vectors may contain one or more selectable marker genes which are wellknown in the art.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.

Host Cells

The host cell may be a bacterium, a yeast or other fungal cell, insectcell, a plant cell, or a mammalian cell, for example.

A transgenic multicellular host organism which has been geneticallymanipulated may be used to produce a polypeptide. The organism may be,for example, a transgenic mammalian organism (e.g., a transgenic goat ormouse line).

Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MN1294 (ATCC 31,446); E. coliX1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P),Pseudomonas such as P. aeruginosa, and Streptomyces. These examples areillustrative rather than limiting. Strain W3110 is one particularlypreferred host or parent host because it is a common host strain forrecombinant polynucleotide product fermentations. Preferably, the hostcell secretes minimal amounts of proteolytic enzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding polypeptides endogenous to the host, with examples of suchhosts including E. coli W3110 strain 1A2, which has the completegenotype tonA; E. coli W3110 strain 9E4, which has the complete genotypetonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has thecomplete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan′; E.coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoAE15 (argF-lac) 169 degP ompT rbs7 ilvG kan′; E. coli W3110 strain 40B4,which is strain 37D6 with a non-kanamycin resistant degP deletionmutation; and an E. coli strain having mutant periplasmic protease.Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

In these prokaryotic hosts, one can make expression vectors, which willtypically contain expression control sequences compatible with the hostcell (e.g., an origin of replication). In addition, any number of avariety of well-known promoters will be present, such as the lactosepromoter system, a tryptophan (trp) promoter system, a beta-lactamasepromoter system, or a promoter system from phage lambda. The promoterswill typically control expression, optionally with an operator sequence,and have ribosome binding site sequences and the like, for initiatingand completing transcription and translation.

Eukaryotic microbes may be used for expression. Eukaryotic microbes suchas filamentous fungi or yeast are suitable cloning or expression hostsfor polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis(MW98-8C, CBS683, CBS4574), K. fragilis (ATCC 12,424), K. bulgaricus(ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris; Candida; Trichoderma reesia;Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans, and A. niger.Methylotropic yeasts are suitable herein and include, but are notlimited to, yeast capable of growth on methanol selected from the generaconsisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,Torulopsis, and Rhodotorula. Saccharomyces is a preferred yeast host,with suitable vectors having expression control sequences (e.g.,promoters), an origin of replication, termination sequences and the likeas desired. Typical promoters include 3-phosphoglycerate kinase andother glycolytic enzymes. Inducible yeast promoters include, amongothers, promoters from alcohol dehydrogenase, isocytochrome C, andenzymes responsible for maltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides as described herein and insome instances are preferred (See Winnacker, From Genes to Clones VCHPublishers, N.Y., N.Y. (1987). For some embodiments, eukaryotic cellsmay be preferred, because a number of suitable host cell lines capableof secreting heterologous polypeptides (e.g., intact immunoglobulins)have been developed in the art, and include CHO cell lines, various Coscell lines, HeLa cells, preferably, myeloma cell lines, or transformedB-cells or hybridomas. In some embodiments, the mammalian host cell is aCHO cell.

In some embodiments, the host cell is a vertebrate host cell. Examplesof useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR(CHO or CHO-DP-12 line); mouse sertoli cells; monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Formulations and Methods of Making the Formulation

Provided herein are also formulations and methods of making theformulation comprising the polypeptides (e.g., antibodies) purified bythe methods described herein. For example, the purified polypeptide maybe combined with a pharmaceutically acceptable carrier.

The polypeptide formulations in some embodiments may be prepared forstorage by mixing a polypeptide having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution.

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some embodiments, the polypeptide in the polypeptide formulationmaintains functional activity.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in addition to a polypeptide, it may be desirable toinclude in the one formulation, an additional polypeptide (e.g.,antibody). Alternatively, or additionally, the composition may furthercomprise a chemotherapeutic agent, cytotoxic agent, cytokine, growthinhibitory agent, anti-hormonal agent, and/or cardioprotectant. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

Exemplary formulations of the anti-IL13 antibodies described herein areprovided in International Patent Pub. No. WO 2013/066866.

Articles of Manufacture

The polypeptides purified by the methods described herein and/orformulations comprising the polypeptides purified by the methodsdescribed herein may be contained within an article of manufacture. Thearticle of manufacture may comprise a container containing thepolypeptide and/or the polypeptide formulation. In certain embodiments,the article of manufacture comprises: (a) a container comprising acomposition comprising the polypeptide and/or the polypeptideformulation described herein within the container; and (b) a packageinsert with instructions for administering the formulation to a subject.

The article of manufacture comprises a container and a label or packageinsert on or associated with the container. Suitable containers include,for example, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds or contains a formulation and may have a sterile access port (forexample the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is the polypeptide. The label orpackage insert indicates that the composition's use in a subject withspecific guidance regarding dosing amounts and intervals of polypeptideand any other drug being provided. The article of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes. In some embodiments, the container is a syringe. In someembodiments, the syringe is further contained within an injectiondevice. In some embodiments, the injection device is an autoinjector.

A “package insert” is used to refer to instructions customarily includedin commercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindications,other therapeutic products to be combined with the packaged product,and/or warnings concerning the use of such therapeutic products.

Exemplary articles of manufacture containing formulations of theanti-IL13 antibodies described herein are provided in InternationalPatent Pub. No. WO 2013/066866.

Further details of the invention are illustrated by the followingnon-limiting Examples. The disclosures of all references in thespecification are expressly incorporated herein by reference.

EXAMPLES

As used in the Examples below and elsewhere herein, “PLB2” and “PLBL2”and “PLBD2” are used interchangeably and refer to the enzyme“phospholipase B-like 2” or its synonym, “phospholipase B-domain-like2”.

Example 1—General Methods

Materials and methods for all Examples were performed as indicated belowunless otherwise noted in the Example.

MAb Feedstocks

MAb feedstocks for all examples were selected from industrial, pilot orsmall scale cell culture batches at Genentech (South San Francisco,Calif., U.S.A.). After a period of cell culture fermentation, the cellswere separated and, in certain instances, the clarified fluid (harvestedcell culture fluid, HCCF) was purified by Protein A chromatography andone or more additional chromatography steps and filtration steps asindicated in the Examples below.

MAb Quantification

The concentration of antibody was determined via absorbance at 280 and320 nm using a UV-visible spectrophotometer (8453 model G1103A; AgilentTechnologies; Santa Clara, Calif., U.S.A.) or NanoDrop 1000 modelND-1000 (Thermo Fisher Scientific; Waltham, Mass., U.S.A.). Speciesother than antibody (i.e. impurities) were too low in concentration tohave an appreciable effect on UV absorbance. As needed, samples werediluted with an appropriate non-interfering diluent in the range of0.1-1.0 absorbance unit. Sample preparation and UV measurements wereperformed in duplicate and the average value was recorded. The mAbabsorption coefficients ranged from 1.42 to 1.645/mg·ml·cm.

Total CHO Host Cell Protein (CHOP) Quantification

An ELISA was used to quantify the levels of the total host cell proteinscalled CHOP. The ELISAs used to detect CHO proteins in products werebased upon a sandwich ELISA format. Affinity-purified polyclonalantibody to CHOP was coated onto a 96-well microtiter plate. Standards,controls, and samples were then loaded in duplicate into separate wells.CHOP, if present in the sample, will bind to the coat antibody(polyclonal anti-CHOP). After an incubation step, anti-CHOP polyclonalantibody-conjugated to horseradish peroxidase (HRP) was added to theplate. After a final wash step, CHOP was quantified by adding a solutionof tetramethyl benzidine (TMB), also available as SUREBLUE RESERVE™ fromKPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md., cat no.53-00-03), which when acted on by the HRP enzyme produces a colorimetricsignal. The optical density (OD) at 450 nm was measured in each well. Afive-parameter curve-fitting program (SOFTMAX® Pro, Molecular Devices,Sunnyvale, Calif.) was used to generate a standard curve, and sampleCHOP concentrations were computed from the standard curve. The assayrange for the total CHOP ELISA was from 5 to 320 ng/ml. CHOPconcentration, in ng/mL, refers to the amount of CHOP in a sample usingthe CHOP standard as a calibrator. CHOP ratio (in ng/mg or ppm) refersto the calculated ratio of CHOP concentration to product concentrationand, in certain instances, was the reported value for the test methods.The Total CHOP ELISA may be used to quantify total CHOP levels in asample but does not quantify the concentration of individual proteins.

Murine Monoclonal Anti-Hamster PLBL2 ELISA Assay

The generation of mouse anti-hamster PLBL2 monoclonal antibodies anddevelopment of an ELISA assay for the detection and quantification ofPLBL2 in recombinant polypeptide preparations using such antibodies isdescribed in U.S. Provisional Patent Application Nos. 61/877,503 and61/991,228. Briefly, the assay is carried out as follows.

Murine monoclonal antibody 19C10 was coated onto a half area 96-wellmicrotiter plate at a concentration of 0.5 μg/mL in carbonate buffer(0.05M sodium carbonate, pH 9.6), overnight at 2-8° C. After coating,the plate was blocked with Blocking Buffer (0.15M NaCl, 0.1M sodiumphosphate, 0.1% fish gelatin, 0.05% polysorbate 20, 0.05% Proclin® 300[Sigma-Aldrich]; also referred to as Assay Diluent) to preventnon-specific sticking of proteins. Standards, controls, and samples werediluted in Assay Diluent (0.15M NaCl, 0.1M sodium phosphate, 0.1% fishgelatin, 0.05% polysorbate 20, 0.05% Proclin® 300 [Sigma-Aldrich]) thenloaded in duplicate into separate wells and incubated for 2 hrs at roomtemperature (22-27° C.). PLBL2, if present in the sample, would bind tothe coat (also referred to herein as capture) antibody. After theincubation step described above, unbound materials were washed awayusing Wash Buffer (0.05% polysorbate 20/PBS [Corning cellgro Cat. No.99-717-CM]) and the 15G11 anti-PLBL2 murine monoclonal antibodyconjugated to biotin was diluted in Assay Diluent to a concentration of0.03125 μg/mL and added to the wells of the microtiter plate.

Biotin conjugation was carried out as follows. A biotinylation kit waspurchased from Pierce Thermo Scientific, (P/N 20217, E-Z LinkNHS-Biotin), and streptavidin-HRP (SA-HRP) from Jackson Immuno Cat. No.016-030-084. Instructions in the Pierce Kit were followed. Briefly, IgGwas dialyzed into PBS, pH 7.4, and biotin was added to the protein andmixed at room temperature for 1 hr. The labeled antibody was thendialized against PBS, pH 7.4 to remove excess biotin, filtered, andprotein concentration determined by A280.

After a 2 hr. incubation step with biotinylated 15G11 at roomtemperature, Streptavidin HRP (1:200,000 dilution in Assay Diluent) wasadded to the microtiter plate wells. After a final wash step with WashBuffer (described above), color was developed (for PLBL2 quantification)by adding a solution of TMB (50 μl/well) (SUREBLUE RESERVE™ from KPL,Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md., cat no.53-00-03) followed by incubation at room temperature for 10-20 minutes.Detection was carried out by assessing optical density (OD) at 450 nm ineach well using a Molecular Devices SpectraMax M5e. A four-parametercurve-fitting program (SoftMax Pro v5.2 rev C) was used to generate astandard curve, and sample PLBL2 concentrations were computed from thelinear range of the standard curve. Values in the linear range of thestandard curve were used to calculate nominal PLBL2 (ng/mg or ppm). Thelinear range was approximately EC₁₀-EC₈₅ or 1.5-40 ng/mL as the rangevaried slightly from plate to plate. Values obtained for PLBL2 usingthis ELISA were comparable to estimates made by other methods (e.g.,LC-MS/MS, polyclonal PLBL2 ELISA or total CHOP ELISA when diluted to theLOQ of the assay

Rabbit Polyclonal Anti-Hamster PLBL2 ELISA Assay

The generation of rabbit anti-hamster PLBL2 polyclonal antibodies anddevelopment of an ELISA assay for the detection and quantification ofPLBL2 in recombinant polypeptide preparations using such antibodies isdescribed in U.S. Provisional Patent Application Nos. 61/877,503 and61/991,228. Briefly, the assay is carried out as follows.

Affinity purified rabbit polyclonal antibody was coated onto a half area96-well microtiter plate at a concentration of 0.5 ug/mL in carbonatebuffer (0.05M sodium carbonate, pH 9.6), overnight at 2-8° C. Aftercoating, the plate was blocked with Blocking Buffer (0.15M NaCl, 0.1Msodium phosphate, 0.1% fish gelatin, 0.05% Polysorbate 20, 0.05%Proclin® 300 [Sigma-Aldrich]) to prevent non-specific sticking ofproteins. Standards, controls, and samples were diluted in Assay Diluent(0.15M NaCl, 0.1M sodium phosphate, 0.1% fish gelatin, 0.05% Polysorbate20, 0.05% Proclin® 300 [Sigma-Aldrich]) then loaded in duplicate intoseparate wells and incubated for 2 hr at room temperature (22-27° C.).PLBL2, if present in the sample, would bind to the coat (also referredto herein as capture) antibody. After the incubation step describedabove, unbound materials were washed away using Wash Buffer (0.05%Polysorbate 20/PBS [Corning Cellgro Cat. No. 99-717-CM]) and theaffinity purified rabbit polyclonal antibody conjugated to horseradishperoxidase (HRP) was diluted in Assay Diluent to a concentration of 40ng/mL and added to the wells of the microtiter plate.

HRP conjugation was carried out as follows. A HRP conjugation kit waspurchased from Pierce Thermo Scientific, (P/N 31489, E-Z Link PlusActivated Peroxidase and Kit). Instructions in the Pierce Kit werefollowed. Briefly, IgG was dialyzed into Carbonate-Bicarbonate buffer,pH 9.4, and EZ-Link Plus Activated Peroxidase was added to the proteinand mixed at room temperature for 1 hr. Sodium cyanoborohydride andQuenching buffer were added subsequently to stabilize the conjugationand quench the reaction. The labeled antibody was then dialyzed againstPBS, pH 7.4, filtered, and protein concentration determined by A280.

After a 2 hr. incubation step with HRP conjugated rabbit polyclonalantibody at room temperature, a final wash step with Wash Buffer(described above) was performed. Afterwards, color was developed (forPLBL2 quantification) by adding a solution of TMB (50 ul/well) (SUREBLUERESERVE™ from KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg,Md., cat no. 53-00-03) followed by incubation at room temperature for10-20 minutes. Detection was carried out by assessing optical density(OD) at 450 nm in each well using a Molecular Devices SpectraMax M5e. Afive-parameter curve-fitting program (SoftMax Pro v5.2 rev C) was usedto generate a standard curve, and sample PLBL2 concentrations werecomputed from the linear range of the standard curve. Values in thelinear range of the standard curve were used to calculate nominal PLBL2(ng/mg or ppm). The quantitative range of the assay was 0.5-50 ng/mL.Values obtained for PLBL2 using this ELISA were comparable to estimatesmade by other methods (e.g., murine monoclonal PLBL2 ELISA, LC-MS/MS ortotal CHOP ELISA when diluted to the LOQ of the assay).

LC-MS/MS Assay

For quantification of PLBL2 by LC-MS/MS, a Waters Acquity H-Class BioUPLC and AB Sciex TripleTOF 5600+ mass spectrometer were used. Samplesand calibration standards (recombinant PLBL2 spiked into a recombinanthumanized monoclonal antibody preparation obtained from a mouse NS0 cellline [the NS0 cell line does not contain hamster PLBL2]) were reducedand digested by trypsin. A total of 40 μg digested sample was injectedonto the UPLC, using a Waters BEH300 C18 column, particle size 1.7 μm. Alinear gradient of acetonitrile was used to elute the peptides, at aflow rate of 300 μl/min and a column temperature of 60° C.

Peptides eluting from the UPLC were introduced to the mass spectrometerby electrospray ionization in positive ionization mode. Ion sourcetemperature was set at 400° C., with an IonSpray voltage of 5500 v. anddeclustering potential of 76 v. A collision energy setting of 32 wasused for the fragmentation of selected peptide ions. The massspectrometer was operated in multiple reaction monitoring highresolution (MRM^(HR)) mode, using four specific PLBL2 peptides and theirfragment ion transitions. The parent ions were selected by thequadrupole mass spectrometer with a mass to charge (m/z) selectionwindow of 1.2 amu. Fragment ions of each parent ion were separated bythe time-of-flight mass spectrometer and selected for quantificationpost data acquisition with a selection window of 0.025 amu.

The concentration of PLBL2 in samples was determined by measuring thespecific signal responses of the four transitions, calibrated by thosefrom the standards in the range of 2-500 ppm using a linear fit. Table 2below shows the list of PLBL2 peptides monitored by LC-MS/MS.

TABLE 2 List of PLBL2 Peptides Monitored by LC-MS/MS. TripleTOF 5600+Scan Cycle Fragment Ion of Scan # Scan Type Peptide Interest Parent m/zFragment m/z 1 TOF MS N/A N/A N/A N/A 2 Product Ion SVLLDAASGQLR +2y8615.3461 817.4163 (SEQ ID NO: 31) 3 Product Ion GLEDSYEGR +2y7 513.2304855.3479 (SEQ ID NO: 32) 4 Product Ion AFIPNGPSPGSR +2y9 600.3120868.4272 (SEQ ID NO: 33) 5 Product Ion VTSFSLAK +2y6 426.7449 652.3665(SEQ ID NO: 34)

Example 2—Improved Purification Process to Reduce Hamster PLBL2

A purification process for CHO-produced anti-IL13 MAb (lebrikizumab) wasestablished to support early stage clinical trials and is referred toherein as the “Initial Process.” The Initial Process employed thefollowing chromatographic steps in order: Protein A affinitychromatography (MABSELECT SURE™) followed by cation exchange (POROS® HS)followed by anion exchange (Q SEPHAROSE™ Fast Flow). Additional virusinactivation and filtration steps were included and a finalultrafiltration-diafiltration (UFDF) step. The final product (drugsubstance) was formulated at a concentration of 125 mg/mL in 20 mMhistidine acetate, 6% sucrose, 0.03% polysorbate 20, pH 5.7.

Using the Total CHOP ELISA Assay (described in Example 1 above), weobserved that in-process intermediates and drug substance purifiedaccording to the Initial Process demonstrated atypicaldilution-dependent behavior resulting in a >20% coefficient of variationacross a normalized series of sample dilutions. This dilution-dependentbehavior is exemplified by the data presented in Table 3 in which eachsuccessive two-fold dilution of anti-IL13 MAb product resulted in higherlevels of CHOP (expressed in ppm) as determined using the Total CHOPELISA. Using sensitive analytical methods, such as LC-MS/MS, wedetermined that a single CHOP species, or HCP, was the cause of thisatypical dilution-dependent behavior. In particular, we established thatthe dilution-dependent behavior on the Total CHOP ELISA was due toantigen excess. Further investigation enabled us to identify the singleHCP as an enzyme, hamster phospholipase B-like 2 (PLBL2). By dilutingthe product samples to the limit of assay quantitation (LOQ), we wereable to estimate the level of PLBL2 in clinical lots of lebrikizumabpurified using the Initial Process and determined that levels as high as300 ppm (300 ng/mg) and above were present.

TABLE 3 Product dilution and CHOP levels. Fold Total CHOP Dilution (ppm)2 0.58 4 1 8 2 16 4 32 7 64 14 128 26 256 49 512 97 1024 147 2048 2284096 314 8192 346

This level of impurity (>300 ppm) of a single CHOP species such as weobserved, is considered undesirable in MAb products intended for humanclinical and/or therapeutic use, particularly late stage clinical trialsand beyond. For example, such levels may be immunogenic whenadministered to human subjects as described in Example 3.

Accordingly, we investigated various modifications to the InitialProcess as briefly outlined below. Based on the results of theseinvestigations, we developed an improved purification process, describedin detail below, and referred to herein as “Improved Process.” Use ofthe Improved Process resulted in purified anti-IL13 MAb (lebrikizumab)product containing substantially reduced levels of PLBL2.

Efforts for modifying the purification process to reduce PLBL2 includedmethods orthogonal to the Initial Process including: precipitation,testing various additives to HCCF, additional column washes, hydrophobicinteraction and mixed mode chromatography. These efforts were informedby use of one or more of the assays described in Example 1 to monitorthe effectiveness of each of the modifications investigated forreduction in total CHOP and/or PLBL2 levels. The various modificationsexplored are described below.

Precipitation of CHOP in HCCF and Protein a Pool with Caprylic Acid

Caprylic acid precipitation has been described previously, including usein the monoclonal antibody industry (Wang et al., BioPharmInternational; Downstream Processing 2010, p 4-10, October2009; Brodskyet al., Biotechnology and Bioengineering, 109(10):2589, 2012) toselectively precipitate impurities from target proteins of interest.Caprylic acid, also known as octanoic acid, is a saturated fatty acidwith eight carbons (formula CH₃(CH₂)₆COOH). Studies were done withanti-IL13 MAb to determine whether precipitation of the harvested cellculture fluid (HCCF) or Protein A pool with caprylic acid would lead toreduced CHOP and/or reduction of dilution-dependent behavior in theTotal CHOP ELISA.

The anti-IL13 MAb starting material for these studies was HCCF andProtein A pools from a 1 kL harvest. 1% (v/v) caprylic acid was added tothe HCCF and varying concentrations of caprylic acid (0%-3% v/v) wereadded to Protein A pools at pH 4.5 or pH 5.0. Samples were mixed for ≥5hours at ambient temperature, 0.2 μm filtered, and diluted with TotalCHOP ELISA diluent for detection and quantification using the Total CHOPELISA. Titer of anti-IL13 MAb in HCCF before and after caprylic acidtreatment was determined using an HPLC titer assay performed accordingto standard methods known in the art.

Treatment of HCCF with 1% v/v caprylic acid reduced CHOP byapproximately 5-fold and resulted in a yield of anti-IL13 MAb of 91%.When Protein A pools were treated with various concentrations ofcaprylic acid, ranging from 0-3% v/v, we observed a loss in yieldof >20% at pH 5.0 and no loss in yield at pH 4.5. When we assessed totalCHOP in these caprylic acid-treated Protein A pools, we found a 2-foldto 3-fold reduction of CHOP (FIGS. 1A and B). However, as also shown inFIGS. 1A and B, dilution-dependence was still present under each of theconditions tested indicating that caprylic acid precipitation was noteffective for addressing the dilution-dependent behavior observed in theTotal CHOP ELISA and would thus not be effective for reducing PLBL2levels in this product.

Additives to HCCF

Previous work by Sisodiya et al., Biotech J. 7:1233 (2012) hasdemonstrated that additives such as guanidine or sodium chloride to HCCFcan reduce the CHOP in the subsequently purified Protein A pools. Asarginine has also been shown to reduce CHOP when utilized as a wash onProtein A columns (Millipore Technical Bulletin, Lit. No. TB1024EN00,Rev. A, December, 2005; Millipore Technical Bulletin, Lit. No. 1026EN00,July, 2006, available at www(dot)Millipore(dot)com), we included it asan additive to HCCF. Various salts, chaotropes, and caprylic acid wereadded to the anti-IL13 MAb HCCF to assess the effectiveness of each forreducing the product and CHOP interaction during capture of product onMABSELECT SURE™ (MSS) protein A chromatography. The additives to HCCFtested were: 0.6M guanidine, 0.6M arginine, 0.6M NaCl,phosphate-buffered saline, and 1% caprylic acid.

Samples that had been treated with each of the HCCF additives weresubjected to Protein A chromatography on MSS. Protein A pools wereadjusted to pH 4.9 and further purified on the POROS® HS cation exchangechromatography step using the Initial Process conditions. Protein Apools and POROS® HS pools were diluted and submitted to the Total ChopELISA. Adjusted Protein A pools were also tested on SEC-HPLC accordingto methods known in the art for the assessment of % aggregate, % variantspecies and the like.

Yields on MABSELECT SURE™ were slightly lower for the runs whereguanidine or arginine was added to HCCF. Of all the additives to HCCFtested, guanidine and arginine were the most effective for reducing CHOPlevels substantially (see Table 4) and appeared to reducedilution-dependence on the Protein A pools (data not shown). Furtherdownstream processing of the Protein A pools on POROS® HS, however,showed CHOP ELISA dilution-dependence remaining in the correspondingPOROS® pools as shown in FIG. 2. Accordingly, the data demonstrate thataddition of guanidine or arginine to HCCF would not be effective forreducing PLBL2 levels in this product.

TABLE 4 HCCF Additives and effect on CHOP. Load Yield Total CHOPAdditive pH (%) (ppm) Control (no additive) 7.4 101 3417 0.6M guanidine7.6 90 892 0.6M arginine 7.1 88 1237 0.6M NaCl 7.7 99 2619 PBS 7.4 982773 1% caprylic acid 6 93 3173

Washing of Protein a Column (MABSELECT SURE™)

It was observed that the more dilution-dependent CHOP eluted in earlyproduct-containing fractions on MABSELECT SURE™ (MSS) Protein Achromatography. This suggested that an additional wash step on MSSbefore elution might further reduce CHOP/PLBL2. Several washes on MSSwere tested for their ability to reduce CHOP/PLBL2 in the Protein Apools. For this study, purified anti-IL13 MAb UFDF pool was used as theload material. The UFDF pool was diluted to 1.7 mg/mL (approximate HCCFtiter) and loaded onto MSS at 29 g/L resin. Various washes were tested,for example; 0.5M arginine pH 8.5, 0.5M arginine pH 9.5 with and without1% polysorbate 20, 0.5M TMAC pH 7.1, 25 mM MOPS pH 7.1, and comparedwith a high salt wash pH 7.0. Product was eluted under acidic conditions(pH 2.8) and pooled beginning at 0.5 OD (A280) and continuing for atotal volume of 2.4 column volumes. Each adjusted pool was diluted andassayed using the Total CHOP ELISA. The summary of these results is thatnone of the washes adequately reduced CHOP/PLBL2 or dilution-dependencein the Total CHOP ELISA. It thus appeared unlikely we would find proteinA wash conditions that would be effective for reducing PLBL2 levels inthis anti-IL13 MAb product and we did not investigate these further.

Washing of Cation Exchange Column (POROS® HS)

Based on theoretical calculations using the amino acid sequences ofanti-IL13 MAb and the PLBL2 impurity, we estimated that the pI of PLBL2is approximately 6.0 and similar to anti-IL13 MAb (pI 6.1). We alsoestimated that there would be a significant difference in net chargebetween anti-IL13 MAb and PLBL2 at pH 4 and pH 10. As such, we testedvarious low pH washes on the Initial Process POROS® HS cation exchangestep to assess whether these would be effective for selectively reducingtotal CHOP and/or PLBL2 and dilution-dependence behavior. The followingwashes were tested at pH 4: (i) acetate gradient, 300 mM-1,000 mM over20 column volumes (CV); (ii) citrate gradient, 100 mM-500 mM over 20 CV;(iii) citrate wash step at 260 mM; and (iv) arginine gradient to 15mS/cm (conductivity measurement) over 20 CV.

The results showed that anti-IL13 MAb and CHOP did not elute with the pH4 acetate gradient up to the tested salt concentration of 1M. Increasingamounts of citrate or acetate resulted in product insolubility andprecipitation. All of the pH 4 washes resulted in low yield on thePOROS® HS step and none of the washes significantly reduced CHOPdilution-dependence. Accordingly, inclusion of a low pH wash of thecation exchange column was not effective for reducing PLBL2 levels inthis product.

Hydroxyapatite Resin and CAPTO™ Adhere Resin

Ceramic hydroxyapatite (CHT) macroporous resin Type I, 40 um (BioRad) iscomprised of calcium phosphate (Ca₅(PO₄)₃OH)₂ in repeating hexagonalstructures. There are two distinct binding sites; C-sites with sets of 5calcium ion doublets and P-sites containing pairs of —OH containingphosphate triplets. This resin has mixed mode properties and has beenshown to separate challenging impurities such as aggregates (P. Gagnon,New Biotechnology 25(5):287 (2009)).

To identify initial conditions for running a CHT column, we performedhigh throughput robot screening of CHT resin Type I, 40 um testing a pHrange of 6.5-8.0 and varying concentrations of sodium chloride andsodium phosphate for elution. Such high throughput robot screenings havebeen previously described, for example, in Wensel et al., Biotechnol.Bioeng. 100:839 (2008). Samples from these screenings were tested in theTotal CHOP ELISA.

CAPTO™ Adhere (GE Healthcare) is a mixed mode resin that exhibits bothionic and hydrophobic properties. The base matrix is a rigid agarose,and the ligand is N-benzyl-N-methylethanolamine. The ability of thisresin to reduce total CHOP and/or PLBL2 was assessed first with ahigh-throughput screening study and then with subsequent columnconditions.

Initial studies to identify conditions for running a CAPTO™ Adherecolumn were done using a high-throughput robot screening method similarto that described above to test binding of anti-IL13 MAb to CAPTO™Adhere at two load densities (5 g/L resin and 40 g/L resin). Salt and pHranges were also tested; from 25 mM-200 mM sodium acetate and pH4.0-6.5. The load material was the Initial Process UFDF pool thatcontained approximately 200 ppm of total CHOP at LOQ by the Total CHOPELISA. Samples of the unbound (flow-through) on CAPTO™ Adhere werediluted and assayed using the Total CHOP ELISA.

The results were as follows. For CHT chromatography, none of the testedconditions substantially reduced total CHOP or PLBL2 or affected assaydilution-dependence behavior. In addition, yields were poor and noclearance of high molecular weight species was achieved. For CAPTO™Adhere chromatography, yields were poor and the assayed material showedsubstantial dilution-dependence behavior in the Total CHOP ELISA.Accordingly, the use of CHT and CAPTO™ Adhere resins were not exploredfurther as it was clear that we would be unlikely to find conditionsusing these resins that would be effective for reducing PLBL2 levels inthis anti-IL13 MAb product.

Hydrophobic Interaction Chromatography Resins and Membranes

We initially tested HIC membrane adsorber referred to as Sartobind andmanufactured by Sartorius. Sartobind is made with a base matrix ofregenerated cellulose and covalently linked hydrophobic phenyl ligandgroups.

The membrane tested was Sartobind HIC 3 mL device (8 mm bed height). Weadjusted the pool from the Initial Process POROS® HS pool to 0.55Mpotassium phosphate pH 7.0 and used a flow rate of 10 mL/min. Productwas eluted in 0.55M potassium phosphate pH 7.0 (collected in the unboundfractions in 3 mL fractions).

We observed that the anti-IL13 MAb became hazy and turbid uponconditioning to 0.55M potassium phosphate and required an additional 0.2um filtration step. The results showed a reduction in total CHOP,however, the remaining CHOP still demonstrated dilution dependentbehavior in the Total CHOP ELISA. Use of this membrane was not evaluatedfurther as it seemed unlikely that effective conditions would beidentified for reducing PLBL2 levels in this product.

Next, we employed a high throughput screen to evaluate several differentHIC resins. OCTYL-SEPHAROSE® Fast Flow (FF), BUTYL-SEPHAROSE® 4 FastFlow, PHENYL SEPHAROSE™ 6 Fast Flow (high sub) and PHENYL SEPHAROSE™ 6Fast Flow (low sub) were obtained from GE Healthcare. These four resinswere chosen because they represent a wide range of varyinghydrophobicity (OCTYL-SEPHAROSE® Fast Flow is the least hydrophobic,followed by PHENYL SEPHAROSE™ 6 Fast Flow (low sub) and BUTYL-SEPHAROSE®4 Fast Flow, with PHENYL SEPHAROSE™ 6 Fast Flow (high sub) the mosthydrophobic. We tested several combinations of pH and saltconcentrations for their effectiveness at reducing PLBL2 in anti-IL13MAb preparations. The anti-IL13 MAb preparation employed for the HICresin experiments was a UFDF pool from a run using the Initial Process.The anti-IL13 MAb concentration was 180 mg/mL and the load density was40 mg antibody/mL resin. We tested pH 5.5 (25 mM sodium acetate), pH 6.0(25 mM MES), pH 7.0 (25 mM MOPS), and pH 8.0 (25 mM Tris) and sodiumsulfate concentrations between 0 mM and 400 mM. For each conditiontested, flow-through samples were collected, diluted and tested usingthe Total CHOP ELISA assay.

The results are shown in FIGS. 3A-D. With increasing salt, we observedless total CHOP in the flow-through for each resin. The OCTYL-SEPHAROSE®Fast Flow resin (FIG. 3A) showed the highest level of total CHOP whilethe PHENYL SEPHAROSE™ 6 Fast Flow (high sub) resin reduced total CHOP tovery low levels, even with lower amounts of salt (FIG. 3D) and thePHENYL SEPHAROSE™ 6 Fast Flow (low sub) and BUTYL-SEPHAROSE® Fast Flowresins showed intermediate levels of total CHOP. Interestingly, therewas also minimal effect of pH on CHOP removal using each of the resinsexcept for PHENYL SEPHAROSE™ 6 Fast Flow (high sub) in low saltconditions (FIG. 3D). For this resin, at low salt conditions, higher pHresulted in higher CHOP in the flow-through fraction (FIG. 3D). Based onthese results, PHENYL SEPHAROSE™ 6 Fast Flow (high sub) appearedpromising and was chosen for further studies which included running thecolumn in either bind-elute or flow-through mode.

Operation of HIC using the PHENYL SEPHAROSE™ 6 Fast Flow (high sub)resin in the bind-elute mode required conditioning of the anti-IL13 MAbload with salt to enable binding of the antibody to the resin.Increasing salt increased the dynamic binding capacity (mg ofanti-IL13/mL resin) for loading product to the resin. But withincreasing salt concentration in the product, we observed increasedturbidity and formation of high molecular weight species (HMWs), inparticular in combination with lower pH.

As mentioned above, PHENYL SEPHAROSE™ 6 Fast Flow (high sub) may also beoperated in flow-through mode and such operation would require less saltconditioning of the load. From a product quality and product stabilityviewpoint, for example, product with less turbidity and less HMWs, lesssalt conditioning would be desirable. Accordingly, we proceeded withprocess optimization using PHENYL SEPHAROSE™ 6 Fast Flow (high sub)resin in flow-through mode.

To optimize the process, we investigated numerous parameters for runningthe HIC column including load concentration, load pH, load saltmolarity, load density on the resin, bed height, flowrate, temperature,equilibration buffer pH and molarity. For these experiments, wemonitored total CHOP using the Total CHOP ELISA and also PLBL2 byLC-MS/MS. Certain exemplary data is shown in Table 5. The data in Table5 shows that the HIC column run under the indicated conditions inflow-through mode was effective for substantially reducing PLBL2 levelsfrom the high levels detected in the Protein A pool. The PLBL2 levelsafter HIC were reduced by several hundred fold compared to the levels inthe Protein A pool.

TABLE 5 Total CHOP and PLBL2 Levels under Varying HIC Column Conditions.Total CHOP PLBL2 Sample (ppm by ELISA (ppm by (bed height, flow rate) %Yield at LOQ) LC-MS/MS) Protein A pool (Load 3324 957 for HIC Column) 15cm, 150 cm/hr 88 43 4 25 cm, 150 cm/hr 92 44 2 15 cm, 100 cm/hr 90 67 525 cm, 100 cm/hr 92 63 3 15 cm, 200 cm/hr 93 62 6 25 cm, 200 cm/hr 90 724 15 cm, 150 cm/hr 54 76 2

Using the PLBL2 LC-MS/MS assay and other typical product quality assays(e.g., SE-HPLC, CE-SDS, iCIEF) to guide process parameter selections, weidentified the following conditions as desirable for running of the HICcolumn as assessed by product quality attributes and reduction of PLBL2:equilibration and wash buffer: 50 mM sodium acetate, pH 5.0; target loaddensity: 100 g/L, flow rate: 150 cm/hr, 22° C.±3° C. Certain smallvariations of these conditions may also be desirable, for example, 25°C.±3° C. or 27° C.±3° C. Optical density (OD) was monitored byabsorbance at 280 nm (A280) and the pool (i.e. the flow-through) wascollected between 0.5 OD to 1.5 OD or after 8 column volumes of wash.

As mentioned above, the Initial Process was: Protein A affinitychromatography (MABSELECT SURE™) followed by cation exchange (POROS® HS)followed by anion exchange (Q SEPHAROSE™ Fast Flow). After developingprocesses to reduce PLBL2 levels as described above, we next sought toimplement process changes in a convenient manner. Accordingly, weexplored adding the HIC column to the Initial Process thereby creating afour-column process as well as substituting the HIC column for eitherthe CEX column or the AEX column and finally we explored the order ofthe columns. We found that a three column process, Protein A affinitychromatography (MABSELECT SURE™), followed by anion exchange (QSEPHAROSE™ Fast Flow), followed by HIC operated in flow-through mode(PHENYL SEPHAROSE™ 6 Fast Flow (high sub)) provided the most convenientprocess and was the most effective for reducing PLBL2 in the final drugsubstance. This three-column process is described in detail below.

The first affinity chromatography step was a bind-and-elute processusing MABSELECT SURE™ resin. After column equilibration (25 mM sodiumchloride, 25 mM Tris pH 7.7), the HCCF was loaded on the column andwashed with the equilibration buffer and a high salt pH 7.0 wash buffer.Anti-IL13 MAb was eluted from the column under acidic conditions (pH2.8).

The second anion-exchange chromatography step was operated in abind-and-elute mode using Q SEPHAROSE™ Fast Flow (QSFF) resin. Aftercolumn equilibration (50 mM Tris, pH 8.0), the anti-IL13 pool from theMABSELECT SURE™ column was adjusted to pH 8.05 and loaded onto thecolumn. The column was washed (50 mM Tris, pH 8.0) and anti-IL13 MAbeluted from the column with 85 mM sodium chloride, 50 mM Tris pH 8.0.

The third and final hydrophobic interaction chromatography step wasoperated in a flow-through mode using PHENYL SEPHAROSE™ 6 Fast Flow(High Sub) resin. After column equilibration (50 mM sodium acetate pH5.0), the anti-IL13 pool from the QSFF column was adjusted to pH 5.0 andloaded on the column. The anti-IL13 MAb flowed through and the columnwas also washed with equilibration buffer (50 mM sodium acetate pH 5.0).The anti-IL13 MAb pool was initiated and terminated based on A280 withpooling occurring between 0.5 to 1.5 OD or a maximum of 8 columnvolumes.

As with the Initial Process, additional virus inactivation andfiltration steps were included and a final ultrafiltration-diafiltration(UFDF) step. The final product (drug substance) was formulated at aconcentration of 125 mg/mL in 20 mM histidine acetate, 6% sucrose, 0.03%polysorbate 20, pH 5.7.

A comparison of the Initial Process to the Improved Process with respectto total CHOP and PLBL2, as measured by the Total CHOP ELISA and themonoclonal PLBL2 ELISA, respectively, is provided in Tables 6 (InitialProcess) and 7 (Improved Process). The data in Table 6 clearly showsthat the Initial Process resulted in purified product (UFDF pool)containing high levels of total CHOP (179, 310, and 189 ng/mg in threedifferent runs) and high levels of PLBL2 (242, 328, and 273 ng/mg inthree different runs) while the data in Table 7 clearly shows that theImproved Process was quite effective for producing purified product withsubstantially reduced levels of total CHOP (1.1, <0.9, 2.8, and 3.4ng/mg in four different runs) and substantially reduced levels of PLBL2(0.21, 0.42, 0.35, and 0.24 ng/mg in four different runs). Consistentwith the data presented above, the data in Table 7 shows that the HICcolumn run under the conditions described above was particularlyeffective for reducing total CHOP and PLBL2 levels in anti-IL13 MAbpreparations.

TABLE 6 Total CHOP and PLBL2 Levels at Various Stages of Purification ofAnti-IL13 MAb Using the Initial Process. In-process sample Total CHOPPLBL2 (ng/mg at LOQ by ELISA) (ng/mg by ELISA) Run No. 1 2 3 1 2 3 HCCF620920 541072 608789 1895 3669 2535 ProA Pool 2892 2855 3505 587 769 478CEX Pool 136 310 138 345 439 287 AEX Pool 104 163 93 291 304 261 UFDFPool 179 310 189 242 328 273

TABLE 7 Total CHOP and PLBL2 Levels at Various Stages of Purification ofAnti-IL13 MAb Using the Improved Process. In-process sample Total CHOPPLBL2 (ng/mg at LOQ by ELISA) (ng/mg by ELISA) Run No. 1 2 3 4 1 2 3 4HCCF 332132 399157 540134 644549 4084 3770 3077 2986 ProA Pool 2318 27683552 3797 1354 1995 1027 975 AEX Pool 495 653 414 377 723 933 677 616HIC Pool <2.1 <1.9 5.0 7.7 <0.6 <0.6 <0.6 <0.6 UFDF 1.1 <0.9 2.8 3.40.21 0.42 0.35 0.24 Pool

In summary, faced with the problem of assay non-linear dilution behaviorattributable to high levels of a single CHOP species in purifiedanti-IL13 MAb preparations, we first identified the CHOP species ashamster PLBL2, an impurity which has not been previously described inrecombinant protein preparations produced from CHO cells. We nextidentified purification conditions to effectively reduce the levels ofPLBL2 in the anti-IL13 MAb preparations. Finally, we integrated thesepurification conditions into the overall purification process resultingin an improvement to the prior anti-IL13 MAb purification process. ThisImproved Process employs a HIC column run in flow-through mode to reducePLBL2 levels, which is run in combination with an affinitychromatography step and an anion exchange chromatography step. We showedthat the Improved Process is robust and effective for substantiallyreducing hamster PLBL2 levels in anti-IL13 MAb preparations. We showedthat the Improved Process reproducibly reduced PLBL2 levels byapproximately 1000 fold compared to the Initial Process. Such reductionin PLBL2 levels was important for producing a purified anti-IL13 MAbproduct suitable for therapeutic use in patients in late stage clinicaltrials and beyond.

Purification Process to Reduce Hamster PLBL2 in Anti-Abeta AntibodyPreparations

We next sought to assess whether the purification methods describedabove, particularly use of a HIC column for a final chromatography step,would similarly be effective for reducing PLBL2 levels in other antibodypreparations. For this experiment, we chose an anti-Abeta antibody,which was produced in CHO cells. Exemplary anti-Abeta antibodies andmethods of producing such antibodies have been described previously, forexample, in WO2008011348, WO2007068429, WO2001062801, and WO2004071408.These particular experiments used the anti-Abeta antibody known ascrenezumab. As described for the anti-IL13 MAb, we explored variousresins and buffers for the second column after the Protein A affinitycolumn and we explored various buffers and run conditions for the HICcolumn to identify those that were optimal for anti-Abeta for productquality and stability attributes as well as for removal of hamsterPLBL2.

We found that a three column process, Protein A affinity chromatography(MABSELECT SURE™), followed by use of a mixed mode resin (CAPTO™Adhere), followed by HIC operated flow-through mode (PHENYL SEPHAROSE™ 6Fast Flow (high sub)) was convenient and effective for reducing PLBL2 inthe final drug substance. This three-column process is described indetail below.

The first affinity chromatography step was a bind-and-elute processusing MABSELECT SURE™ resin similar to that described above for theanti-IL13 MAb.

The second mixed mode chromatography step was operated in a flow-throughmode using CAPTO™ Adhere resin. After column equilibration (20 mM MES,150 mM sodium acetate, pH 6.25), the anti-Abeta pool from the MABSELECTSURE™ column was adjusted to pH 6.25 and loaded onto the column. Poolingbegan at 0.5 OD during the load phase. After completing the load, thecolumn was washed with 5 column volumes (CVs) of equilibration buffer(20 mM MES, 150 mM sodium acetate, pH 6.25) and the entire 5 CVs werealso collected.

The third and final hydrophobic interaction chromatography step wasoperated in a flow-through mode using PHENYL SEPHAROSE™ 6 Fast Flow(High Sub) resin. After column equilibration (150 mM sodium acetate pH5.0), the anti-Abeta pool from the CAPTO™ Adhere column was adjusted topH 5.0 and loaded on the column. The anti-Abeta MAb flowed through andthe column was also washed with equilibration buffer (150 mM sodiumacetate pH 5.0). The anti-Abeta MAb pool was initiated during the loadphase based on A280 with pooling beginning at 0.5 OD. The column waswashed with 10 CVs of equilibration buffer (150 mM sodium acetate pH5.0) and the entire 10 CVs were also collected. As with the anti-IL13MAb, additional virus inactivation and filtration steps were includedand a final ultrafiltration-diafiltration (UFDF) step.

The results of using the above process during four differentpurification runs are shown in Table 8 below.

TABLE 8 PLBL2 Levels at Various Stages of Purification of Anti-Abeta MAbUsing HIC. In-process sample PLBL2 (ng/mg by ELISA) Run No. 1 2 3 4 HCCF622 564 1264 553 CpA Pool 7 8 9 2.5 HIC Pool 0.7 0.6 0.3 0.3 (300 g/LLoad density) HIC Pool <0.2 <0.2 <0.2 Not (100 g/L tested Load density)

The results shown in Table 8 demonstrate that use of a HIC resin as afinal chromatography step effectively reduced residual PLBL2 levels inthe anti-Abeta MAb preparation to an amount similar to that seen for theanti-IL13 MAb. While a load density of 300 g/L produced desirableresults from the viewpoint of both product recovery and reduction inPLBL2, further reduction in residual PLBL2 was seen by reducing the loaddensity for the HIC column from 300 g/L to 100 g/L.

We also investigated two other conditions for the HIC chromatographystep, load pH and load sulfate molarity. For these experiments, westarted with a CAPTO′ Adhere pool containing 51 ng/mg PLBL2 (as measuredby ELISA), 15 mM sodium acetate pH 5.5. We adjusted the load pH and theload sulfate molarity to the values shown in Table 9 below using 0 mMsodium sulfate or 800 mM sodium sulfate stock solutions at varying pH.We tested each load pH indicated in Table 9 under low sulfate molarityconditions (0 mM) and high sulfate molarity conditions (240 mM). Eachcondition was tested at a load density of 60 g/L. As shown by theresults presented in Table 9, decreasing the load pH to pH 4 or pH 5 orincreasing the load sulfate molarity (to 240 mM sulfate) were eacheffective for reducing the levels of PLBL2 in the final HIC pool. Thecombination of pH 4.0 and 240 mM sulfate in the load was particularlyeffective for minimizing the amount of residual PLBL2 in the HIC pool.

TABLE 9 PLBL2 levels in the HIC pool observed over a range of load pHand sulfate molarity. PLBL2 (ng/mg by ELISA) Load Low Sulfate MolarityHigh Sulfate Molarity pH (0 mM) (240 mM) 4 4 1 5 10 3 6 27 5 7 64 6

Accordingly, use of a HIC resin as a final chromatography step in thepurification of CHO-produced polypeptides, such as the anti-IL13 MAb andthe anti-Abeta MAb described herein, effectively reduced the residualamount of hamster PLBL2 to very low levels, e.g., 1 ng/mg or less in theHIC pool.

Purification Process to Reduce Hamster PLBL2 in IgG1 AntibodyPreparations

Next, we assessed whether the purification methods described for theanti-IL13 and anti-Abeta IgG4 antibody preparations, particularly use ofa HIC column for a final chromatography step, would similarly beeffective for reducing PLBL2 levels in IgG1 antibody preparations. Forthese experiments, we first chose an anti-IL17 A/F antibody, which is anIgG1 antibody and which was produced in CHO cells. Exemplary anti-IL17A/F antibodies and methods of producing such antibodies have beendescribed previously, for example, in WO 2009136286 and U.S. Pat. No.8,715,669. As described for the anti-IL13 and anti-Abeta MAbs, weexplored various resin (in particular, PHENYL SEPHAROSE™ FF [low sub]and PHENYL SEPHAROSE™ FF [high sub] and buffer conditions (inparticular, 50 mM sodium acetate, pH 5.5 and 50 mM Tris, 85 mM sodiumacetate, pH 8.0) for the HIC column to identify those that were optimalfor anti-IL17 A/F for product quality and stability attributes as wellas for removal of hamster PLBL2.

We found that a three column process, Protein A affinity chromatography(MABSELECT SURE™), followed by cation exchange chromatography (POROS®50HS) operated in bind-and-elute mode, and HIC (PHENYL SEPHAROSE™ 6 FastFlow (high sub)) operated in flow-through mode was convenient andeffective for reducing PLBL2 in the final drug substance. Thisthree-column process is described in detail below.

The first affinity chromatography step was a bind-and-elute processusing MABSELECT SURE™ resin similar to that described above for theanti-IL13 and anti-Abeta MAbs. The second cation exchange chromatographystep used POROS® 50HS resin and was operated in bind-and-elute mode.After column equilibration (40 mM sodium acetate, pH 5.5), thepH-adjusted anti-IL17 A/F MABSELECT SURE™ pool (pH 5.0) was loaded ontothe column. The column was washed (40 mM sodium acetate, pH 5.5), andthen the anti-IL17 A/F antibody was eluted from the column with aconductivity gradient created with 40 and 400 mM sodium acetate, pH 5.5.Pooling was based on A280 and was initiated at ≥0.5 OD and ended at ≤2.0OD during the gradient elution phase.

The third and final hydrophobic interaction chromatography step usedPHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin and was operated in aflow-through mode. After column equilibration (50 mM sodium acetate pH5.5), the anti-IL17 A/F pool from the POROS® 50HS column was loadeddirectly on the column without pH adjustment. The anti-IL17 A/F MAbflowed through. Anti-IL17 A/F MAb pooling was based on A280 and wasinitiated during the load phase at ≥0.5 OD. The column was washed with10 CVs of equilibration buffer (50 mM sodium acetate, pH 5.5) andpooling ended during this wash phase at ≤1.0 OD.

The results of using the above process during one purification run areshown in Table 10 below.

TABLE 10 PLBL2 Levels at Various Stages of Purification of Anti-IL17 A/FMAb Using HIC. In-Process PLBL2 Sample (ng/mg by ELISA) Run No. 1 HCCF713 MABSELECT 151 SURE ™ Pool POROS ® 50HS Pool 47 HIC Pool <0.7 (100g/L load density)

The results shown in Table 10 demonstrate that use of a HIC resin as afinal chromatography step effectively reduced residual PLBL2 levels inthe anti-IL17 A/F MAb (IgG1) preparation to an amount similar to thatseen for the anti-IL13 and anti-ABeta MAbs (IgG4).

Anti-CMV Antibody

In addition to testing anti-IL17 A/F, we tested another IgG1 MAb,anti-CMV-MSL antibody, which is also produced in CHO cells. Exemplaryanti-CMV antibodies, including anti-CMV-MSL, and methods of producingsuch antibodies have been described previously, for example, in WO2012047732.

Again, we found that a three column process, Protein A affinitychromatography (MABSELECT SURE™), followed by cation exchangechromatography (POROS® 50HS) operated in bind-and-elute mode, and HIC(PHENYL SEPHAROSE™ 6 Fast Flow (high sub)) operated in flow-through modewas convenient and effective for reducing PLBL2 in the final drugsubstance. This three-column process is described in detail below.

The first affinity chromatography step was a bind-and-elute processusing MABSELECT SURE™ resin similar to that described above for theanti-IL13, anti-Abeta and anti-IL17 A/F MAbs. The second cation exchangechromatography step that used POROS® 50HS resin and was operated inbind-and-elute mode. After column equilibration (40 mM sodium acetate,pH 5.5), the pH-adjusted aCMV-MSL MABSELECT SURE™ pool (pH 5.0) wasloaded onto the column. The column was washed (40 mM sodium acetate, pH5.5), and then the aCMV-MSL antibody was eluted from the column with aconductivity gradient created with 40 and 400 mM sodium acetate, pH 5.5.Pooling was based on A280 and was initiated at ≥0.5 OD and ended at ≤1.0OD during the gradient elution phase.

In this particular run, a viral filtration step was performed in betweenthe cation exchange and hydrophobic interaction chromatography stepsusing Viresolve Pro as the virus filter and Fluorodyne UEDF filter asthe pre-filter.

The third and final hydrophobic interaction chromatography step usedPHENYL SEPHAROSE™ 6 Fast Flow (High Sub) resin and was operated in aflow-through mode. After column equilibration (50 mM sodium acetate pH5.5), the anti-CMV-MSL pool from the POROS® 50HS column was loadeddirectly on the column without pH adjustment. The anti-CMV-MSL MAbflowed through. Anti-CMV-MSL MAb pooling was based on A280 and wasinitiated during the load phase at ≥0.5 OD. The column was washed with10 CVs of equilibration buffer (50 mM sodium acetate, pH 5.5) andpooling ended during this wash phase at ≤0.5 OD.

The results of using the above process during one purification run isshown in Table 11 below.

TABLE 11 PLBL2 Levels at Various Stages of Purification of Anti-CMV-MSLMAb Using HIC. In-Process PLBL2 Sample (ng/mg by ELISA) Run No. 1 HCCF2608 MABSELECT 319 SURE ™ Pool POROS ® 50HS Pool 33 Viresolve Pro Pool32 HIC Pool <0.6 (60 g/L load density)

The results shown in Table 11 demonstrate that use of a HIC resin as afinal chromatography step effectively reduced residual PLBL2 levels inthe anti-CMV-MSL MAb preparation to an amount similar to that seen forthe anti-IL13, anti-ABeta, and anti-IL17 A/F MAbs. Accordingly, use of aHIC resin as a final chromatography step in the purification ofCHO-produced polypeptides, such as the anti-IL13 MAb and other MAbsdescribed herein, effectively reduced the residual amount of hamsterPLBL2 to very low levels, e.g., less than 1 ng/mg in the HIC pool. Thus,we showed that use of the HIC chromatography step as described hereinfor reducing PLBL2 levels was as effective for IgG1 MAbs as for IgG4MAbs, illustrating the general applicability of this method for reducinghamster PLBL2 levels in recombinant polypeptide preparations.

Example 3—Assessment of Human Anti-Hamster PLBL2 Response in PatientsAdministered Anti-IL13 MAb Compositions Containing Varying Amounts ofHamster PLBL2

To assess the potential impact of the CHO PLBL2 impurity, we developedan ELISA assay (a bridging ELISA assay) to detect antibodies to hamsterPLBL2 in human subjects who had received the anti-IL13 MAb,lebrikizumab. Serum samples from patients who participated in variousclinical studies of lebrikizumab were analyzed for evidence ofanti-hamster PLBL2 antibodies pre-dose and post-dose as well as insubjects who received placebo. The details of the clinical studies havebeen described previously (WO 2012/083132, Corren et al., N Engl J Med365:1088-98 (2011)) and only the most relevant details of these studiesare provided below.

The antibody bridging ELISA assay that was developed and validated todetect antibodies to hamster PLBL2 in human serum used two conjugatedreagents to capture all isotypes of antibodies directed against hamsterPLBL2: purified hamster PLBL2 conjugated to biotin (Biotin-PLBL2) andpurified hamster PLBL2 conjugated to digoxigenin (DIG-PLBL2). Productionand purification of hamster PLBL2 was carried out using standard methodsknown to one skilled in the art is also described in U.S. ProvisionalApplication Nos. 61/877,503 and 61/991,228 and conjugation to biotin orDIG were carried out using standard methods known to one skilled in theart. In this semi-homogenous antibody bridging ELISA assay, 75 μL/wellof conjugated solution in assay diluent (PBS/0.5% BSA/0.05% Polysorbate20/0.05% ProClin 300, pH 7.4±0.1) containing 3 μg/mL of eachBiotin-PLBL2 and DIG-PLBL2 were co-incubated overnight (16-24 hours) atambient temperature with 75 μL/well of 1:20 diluted serum samples andcontrols in assay diluent in polypropylene micronic tubes (NationalScientific Supply Co.; Claremont, Calif.). After incubation, 100 μL/wellof mixture from the micronic tubes were transferred to astreptavidin-coated 96-well microplate (StreptaWell™ High Bind; RocheDiagnostics; Indianapolis, Ind.) that was washed 3 times with 400μL/well of wash buffer (PBS/0.05% Polysorbate 20) in an automatic platewasher (BioTek ELx405) and incubated at ambient temperature for 2 hours±10 minutes. The plate was washed 4 times with 400 μL/well of washbuffer in the plate washer, Subsequently, 100 μL/well of 400 ng/mL mouseanti-digoxin antibody conjugated with horseradish peroxidase (HRP)(Jackson ImmunoResearch Cat.200-032-156) was added and incubated atambient temperature for 2 hours±10 minutes for detection. After theplate was washed 4 times with 400 μL/well of wash buffer in the platewasher, 100 μL/well of equal mixture solution of peroxidase substrate(tetramethyl benzidine) (0.4 g/L TMB) and Peroxidase Solution B (0.02%hydrogen peroxide) (KPL Cat. 50-76-03) was added and incubated atambient temperature for 18-28 minutes for color development and thereaction was stopped by adding 100 μL/well of 1 M phosphoric acid. Theplates were read at 450 nm for detection absorbance and 630 nm forreference absorbance. The positive control for the assay was amonoclonal antibody construct consisting of a murine anti-hamsterPLBL2-specific complementarity determining region (CDR) on a human IgG1framework. The relative sensitivity of the assay using this antibody wasdetermined to be 25 ng/mL. Assay drug tolerance experiments using thisantibody demonstrated that up to 50 μg/mL of lebrikizumab or 1 μg/mL ofhamster PLBL2 in serum did not cause interference or cross-reactivity inthe assay.

To carry out the assay, serum samples were first screened in the assayat a minimum dilution of 1/20. Samples that screened positive were thenconfirmed for hamster PLBL2 specificity using a competition confirmatoryassay. If the sample was confirmed as positive, it was serially dilutedto obtain a titer value. Positive responses were reported in titerunits, which is the log 10 of the dilution factor at which the samplesignal was equal to the signal of the assay cutpoint (threshold fordetermining positivity).

The four clinical studies in which patient samples were analyzed usingthe anti-hamster PLBL2 ELISA described above are briefly described asfollows. Study 1 was a Phase II randomized, double-blind,placebo-controlled, proof-of-concept study to evaluate the effects oflebrikizumab in patients with asthma whose disease was inadequatelycontrolled during chronic therapy with inhaled corticosteriods (ICS). Atotal of 219 patients were randomized, with 106 receiving at least one250 mg subcutaneous (SC) dose of lebrikizumab and 92 receiving sixmonthly doses.

Study 2 was a Phase II randomized, double-blind, placebo-controlled,dose-ranging study in patients with asthma who were not on ICS therapy.Patients received one of three doses (500, 250, or 125 mg) oflebrikizumab or placebo via SC administration. Study drug wasadministered four times during the 12-week treatment period. A total of158 patients were exposed to at least one dose of lebrikizumab, and 145patients received all four doses.

Study 3 was a Phase I PK study of lebrikizumab in healthy Japanese andCaucasian volunteers. Three discrete cohorts of 20 healthy Japanese andCaucasian subjects (10 subjects in each racial group) were randomizedbetween lebrikizumab (125, 250, and 375 mg SC) and placebo in a 7:3ratio. Subjects were dosed once on Day land were subsequently monitoredfor 120 days. A total of 42 subjects each received one dose oflebrikizumab.

In Studies 1-3, a total of 306 subjects, 264 of which were asthmapatients, each received at least one dose of material containing hamsterPLBL2. Exposure to hamster PLBL2 was variable, depending on the dose oflebrikizumab received.

Study 4 was a Phase IIb randomized, double-blind, placebo-controlledstudies to assess the efficacy and safety of lebrikizumab in patientswith uncontrolled asthma who were using ICS and a second controllermedication. Patients received one of three doses (250, 125, or 37.5 mg)of lebrikizumab or placebo via SC administration monthly. In Study 4, atotal of 463 patients were randomized, with 347 receiving at least onedose of lebrikizumab. Exposure to hamster PLBL2 was variable, dependingon the dose of lebrikizumab received.

Table 12 below provides a summary of each of the Studies 1-4 showing therange of hamster PLBL2 levels the subjects were exposed to and the doseof lebrikizumab.

TABLE 12 Hamster PLBL2 Exposure in Lebrikizumab Clinical Trials. DrugSubstance PLBL2 Lebrikizumab Dose PLBL2 Study (ng/mg) (mg/month)(μg/dose) 1 34-137^(a) 250 9-34 2 34-137^(a) 125 4-17 250 9-34 50017-69  3  34 125 4 250 9 375 13 4 242 37.5 9 328 125 41 328 250 82^(a)Range from four different lots of clinical material.

A retrospective analysis of selected time points from Study 1 wasperformed using the anti-hamster PLBL2 antibody assay described above todetect antibodies to hamster PLBL2. Samples from both placebo and dosedsubjects were analyzed to determine the level of pre-existing responseas well as the development of antibodies in response to lebrikizumabdosing. There were 113 placebo subjects and 106 dosed subjects whoreceived at least one dose of lebrikizumab. Timepoints selected foranalysis were Days 0, 29, 85, 141, 225, and early termination. Sampleswere taken prior to the next dose; therefore, Day 29 samples were takenprior to the administration of the second dose. The percentage ofanti-hamster PLBL2 antibody-positive subjects at each timepoint wascalculated by taking the number of positive subjects at each timepointand dividing by the total number of subjects tested at each timepoint.The data is shown in Table 13.

TABLE 13 Anti-Hamster PLBL2 Antibody Results for Study 1. % Positive atEach Timepoint (no. positive subjects/total no. subjects evaluable)Study Day: Early 0 29 85 141 225 Termination Placebo 6 7  9  8  5  25(7/110) (8/107)  (9/104)  (8/99)  (5/97) (2/8) 250 mg 5 6 89 98 98 100dose (5/102) (6/100) (90/101) (92/94) (91/93)  (8/8)^(a) ^(a)Of the 8lebrikizumab subjects who discontinued study drug early, only 3 reportedadverse events as the reason for study drug discontinuation.

The 6 Study 1 placebo subjects who were positive pre-dose on Day 0continued to be positive throughout the study. Samples from thesesubjects were confirmed as positive in a confirmatory competition assayand had titers on Day 0 that ranged from 1.6 to 2.9 titer units. Titersobtained on subsequent visits were similar to those obtained on Day 0. Afew additional placebo subjects had low-level positive responses duringthe Study.

Among the Study 1 subjects that received lebrikizumab, 98% (104/106) hada positive antibody response after dosing and remained positive throughthe end of the study, with most subjects becoming positive afterreceiving at least two doses of lebrikizumab. Titers after dosing rangedfrom 1.35 to 4.76 titer units, with titers generally increasing overtime. The clinical significance of the development of anti-hamster PLBL2antibodies is not known. No clinically important safety signals wereidentified in this study and, given the high incidence of antibodies tohamster PLBL2, no correlation with safety events could be made.

An interim analysis was also performed on samples collected in Study 4.Samples from both placebo and dosed subjects were analyzed to determinethe level of pre-existing response as well as the development ofanti-hamster PLBL2 antibodies in response to lebrikizumab dosing. Therewere 116 placebo subjects and 347 dosed subjects who received at leastone dose of lebrikizumab. Samples from 92 placebo subjects and 268 dosedsubjects are represented in this data set. The results are shown inTable 14.

TABLE 14 Anti-Hamster PLBL2 Antibody Results for Study 4 for Subjectsnot Previously Exposed to Lebrikizumab. % Positive at Each Timepoint(no. positive subjects/total no. subjects evaluable) Study Day: Early 029 85 169 253 Termination Placebo 4  4  4  0 NA 0 (4/89) (3/78)  (2/48) (0/13) (0/5)  37.5 mg 9  9 55  79 66 43 dose (8/88) (7/82) (35/64)(27/34) (2/3) (3/7)^(a) 125 mg 4 11 87 100 NA 0 dose (3/81) (8/73)(48/55) (9/9) (0/2)^(a) 250 mg 5 10 96 100 NA 67 dose (4/88) (7/72)(49/51) (13/13) (2/3)^(a) ^(a)Of the 12 lebrikizumab subjects whodiscontinued study drug early, only 4 reported adverse events as thereason for study drug discontinuation.

The four Study 4 placebo subjects that were positive pre-dose on Day 0had low-level positive responses that were just above the detectionlimit of the assay. The low-level responses were detectable at some, butnot all, subsequent timepoints.

The 15 Study 4 subjects receiving lebrikizumab that were positivepre-dose on Day 0 continued to be positive at subsequent timepoints,with increasing titers after multiple doses. In addition, there were 10subjects in Study 4 who previously received lebrikizumab in Study 1.Nine of these subjects were subsequently re-dosed with lebrikizumab inStudy 4 while 1 subject received placebo. All 10 subjects were pre-dosepositive on Day 0 for Study 4 and continued to be positive at subsequenttimepoints. The data from these 10 subjects were excluded from Table 14due to their previous lebrikizumab exposure.

Among the Study 4 subjects receiving lebrikizumab, there appear to bedifferences in positivity rates between dose groups. However, as thesedata are incomplete, conclusions regarding the significance of thesedifferences cannot be made at this time. Similar to the data from Study1, the majority of subjects become positive after receiving at least twodoses of lebrikizumab. Titers after dosing ranged from 1.68 to 4.55titer units, with titers generally increasing over time. Since this isan incomplete data set, positive percentages and titer ranges may changeas additional data are accumulated.

An interim safety assessment of Study 4 showed a safety profile similarthose of the earlier completed studies with no clinically significantsafety signals, including no reports of anaphylaxis or serioushypersensitivity reactions. Of note, 6 of the 9 patients who receivedlebrikizumab in Study 1 and were subsequently re-dosed with lebrikizumabin Study 4 had not reported any adverse events at the time of theinterim analysis and only 1 patient reported any local injection-sitereactions. No clinical sequelae of this anti-hamster PLBL2 antibodyresponse have been identified in the clinical trials to date.

We also performed an assessment on the 125-mg dose group from Study 2and those results are shown in Table 15.

TABLE 15 Anti-Hamster PLBL2 Antibody Results for Study 2. % Positive atEach Timepoint (no. positive subjects/total no. subjects evaluable)Study Day: Early 0 29 57 85 141 Termination 125 mg 4 21 70 88 86 100dose (2/51) (11/53) (35/50) (45/51) (43/50) (2/2)^(a) ^(a)Of the 2subjects who discontinued study drug early, neither reported adverseevents as the reason for study drug discontinuation.

The two Study 2 subjects that were positive pre-dose on Day 0 continuedto be positive at all subsequent timepoints, with increasing titersafter multiple doses. Among the Study 2 subjects that received 125 mg oflebrikizumab, 87% ( 46/53) had a positive antibody response after dosingand remained positive through the end of the study, with most subjectsbecoming positive after receiving at least two doses of lebrikizumab.Titers after dosing ranged from 1.51 to 4.09 titer units, with titersgenerally increasing over time.

CONCLUSIONS

To assess the potential impact of the CHO PLBL2 impurity, an assay wasdeveloped to detect antibodies to hamster PLBL2 in subjects who hadreceived lebrikizumab preparations that contained significant levels ofhamster PLBL2. On the basis of the completed data sets from Study 1 andthe 125 mg dose group of Study 2 and on the partial data set from Study4, the presence of hamster PLBL2 in lebrikizumab preparations producedimmune responses in most subjects exposed to hamster PLBL2.

A number of subjects in both the placebo and lebrikizumab dose groupshad pre-existing immunoreactivity in the anti-hamster PLBL2 antibodyassay. The cause of this pre-existing response is unknown; antibodyreactivity to CHO host cell proteins has previously been characterizedand confirmed in normal human serum samples with no known prior exposureto CHO-derived biological products (Xue et al., The AAPS Journal12(1):98-106 (2010)). However, there are no published data specific tothe single species of CHOP, PLBL2.

For subjects with pre-existing immunoreactivity in the anti-hamsterPLBL2 antibody assay at the start of the study, there was a sustainedrise in antibody titers after repeat administration with lebrikizumab.For subjects that were antibody negative at the start of the study, themajority of subjects across all four studies became positive after atleast two administrations of lebrikizumab and remained positive throughall subsequent timepoints.

The clinical significance of the development of anti-hamster PLBL2antibodies is not known. Although there was a high incidence ofantibodies to hamster PLBL2 in the study subjects, no correlationbetween safety events could be made. Importantly, there were no safetysignals identified in these completed or interim studies and inparticular, no reported events of anaphylaxis, anaphylactoid, or serioushypersensitivity reactions. Nevertheless, there remains a concern thatlong term exposure with repeat dosing could increase the potential forundesirable effects such as anaphylaxis, hypersensitivity, and immunecomplex deposition, particularly in asthma patient populations and otherallergic or hypersensitive patient populations. Accordingly, it isimportant to dose patients in late stage clinical studies and beyond,where there may be such repeat dosing over a long period of time, withanti-IL13 MAb (e.g., lebrikizumab) preparations containing substantiallyreduced levels of hamster PLBL2 so as to minimize immunogenicity as muchas possible.

Additional antibody sequences are provided in Table 16 below.

TABLE 16Anti-IL17 A/F antibody amino acid sequences (SEQ ID NOS.: 15-22) and anti-Abetaantibody amino acid sequences (SEQ ID NOS.: 23-30). CDR-H1Asp Tyr Ala Met His (SEQ ID NO.: 15) CDR-H2Gly Ile Asn Trp Ser Ser Gly Gly Ile Gly Tyr Ala Asp Ser Val (SEQ IDLys Gly NO.: 16) CDR-H3Asp Ile Gly Gly Phe Gly Glu Phe Tyr Trp Asn Phe Gly Leu (SEQ ID NO.: 17)CDR-L1 Arg Ala Ser Gln Ser Val Arg Ser Tyr Leu Ala (SEQ ID NO.: 18)CDR-L2 Asp Ala Ser Asn Arg Ala Thr (SEQ ID NO.: 19) CDR-L3Gln Gln Arg Ser Asn Trp Pro Pro Ala Thr (SEQ ID NO.: 20) VHGlu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg (SEQ IDSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr NO.: 21)Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValSer Gly Ile Asn Trp Ser Ser Gly Gly Ile Gly Tyr Ala Asp Ser ValLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu TyrLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr CysAla Arg Asp Ile Gly Gly Phe Gly Glu Phe Tyr Trp Asn Phe Gly LeuTrp Gly Arg Gly Thr Leu Val Thr Val Ser Ser VLGlu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly (SEQ IDGlu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Tyr NO.: 22)Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu IleTyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu ProGlu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro ProAla Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys CDR-H1 GFTFSSYGMS(SEQ ID NO.: 23) CDR-H2 SINSNGGSTY YPDSVK SEQ ID NO.: 24) CDR-H3 GDYSEQ ID NO.: 25) CDR-L1 RSSQSLVYSN GDTYLH (SEQ ID NO.: 26) CDR-L2 KVSNRFS(SEQ ID NO.: 27) CDR-L3 SQSTHVPWT (SEQ ID NO.: 28) VHEVQLVESGGG LVQPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLELVAS INSNGGSTYY(SEQ IDPDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCASGD YWGQGTTVTV SSASTKGPSVNO.: 29)FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTVLHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCLVKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVMHEALHNHYTQ KSLSLSLG VLDIVMTQSPLS LPVTPGEPAS ISCRSSQSLV YSNGDTYLHW YLQKPGQSPQ LLIYKVSNRF(SEQ IDSGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHVP WTFGQGTKVE IKRTVAAPSVNO.: 30)FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSLSSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

1-93. (canceled)
 94. A method of treating an IL-13-mediated disorder ina patient comprising administering a treatment composition to thepatient, wherein the treatment composition comprises a compositioncomprising an anti-IL13 monoclonal antibody purified from Chinesehamster ovary host cells, wherein the composition comprises theanti-IL13 antibody and a residual amount of hamster PLBL2 of less than20 ng/mg.
 95. The method of claim 94, wherein administration of thetreatment composition is less immunogenic for hamster PLBL2 compared toadministration of a reference composition, wherein the referencecomposition comprises an anti-IL13 monoclonal antibody purified fromChinese hamster ovary host cells and a residual amount of hamster PLBL2of greater than.
 96. The method of claim 94, wherein the treatmentcomposition is administered subcutaneously once every four weeks, onceevery eight weeks, or once every 12 weeks.
 97. The method of claim 96,wherein the patient is treated once every four weeks for at least onemonth.
 98. The method of claim 94, wherein the IL-13-mediated disorderis selected from asthma, idiopathic pulmonary fibrosis and atopicdermatitis.
 99. The method of claim 94, wherein the IL-13-mediateddisorder is selected from allergic asthma, non-allergic asthma, allergicrhinitis, allergic conjunctivitis, eczema, urticaria, food allergies,chronic obstructive pulmonary disease, ulcerative colitis, RSVinfection, uveitis, scleroderma, and osteoporosis.
 100. The method ofclaim 94, wherein the anti-IL13 antibody comprises three heavy chainCDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 1, CDR-H2having the amino acid sequence of SEQ ID NO.: 2, and CDR-H3 having theamino acid sequence of SEQ ID NO.: 3, and three light chain CDRs, CDR-L1having the amino acid sequence of SEQ ID NO.: 4, CDR-L2 having the aminoacid sequence of SEQ ID NO.: 5, and CDR-L3 having the amino acidsequence of SEQ ID NO.:
 6. 101. The method of claim 100, wherein theanti-IL13 antibody comprises a heavy chain variable region having theamino acid sequence of SEQ ID NO.:
 7. 102. The method of claim 100,wherein the anti-IL13 antibody comprises a light chain variable regionhaving the amino acid sequence of SEQ ID NO.:
 9. 103. The method ofclaim 101, wherein the anti-IL13 antibody comprises a heavy chain havingthe amino acid sequence of SEQ ID NO.:
 10. 104. The method of claim 102,wherein the anti-IL13 antibody comprises a light chain having the aminoacid sequence of SEQ ID NO.:
 14. 105. The method of claim 100, whereinthe anti-IL13 antibody comprises a heavy chain variable region havingthe amino acid sequence of SEQ ID NO.: 7 and a light chain variableregion having the amino acid sequence of SEQ ID NO.:
 9. 106. The methodof claim 105, wherein the anti-IL13 antibody comprises a heavy chainhaving the amino acid sequence of SEQ ID NO.: 10 and a light chainhaving the amino acid sequence of SEQ ID NO.:
 14. 107. The method ofclaim 94, wherein the residual amount of hamster PLBL2 is less than 15ng/mg.
 108. The method of claim 94, wherein the residual amount ofhamster PLBL2 is less than 10 ng/mg.
 109. The method of claim 94,wherein the residual amount of hamster PLBL2 is less than 8 ng/mg. 110.The method of claim 94, wherein the residual amount of hamster PLBL2 isless than 5 ng/mg.
 111. The method of claim 94, wherein the residualamount of hamster PLBL2 is less than 3 ng/mg.
 112. The method of claim94, wherein the residual amount of hamster PLBL2 is less than 2 ng/mg.113. The method of claim 94, wherein the residual amount of hamsterPLBL2 is less than 1 ng/mg.
 114. The method of claim 94, wherein theresidual amount of hamster PLBL2 is less than 0.5 ng/mg.
 115. The methodof claim 95, wherein the residual amount of hamster PLBL2 in thereference composition is greater than 200 ng/mg.
 116. The method ofclaim 95, wherein the residual amount of hamster PLBL2 in the referencecomposition is greater than 300 ng/mg.